WO2004067737A2 - Subtilases - Google Patents

Subtilases Download PDF

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Publication number
WO2004067737A2
WO2004067737A2 PCT/DK2004/000066 DK2004000066W WO2004067737A2 WO 2004067737 A2 WO2004067737 A2 WO 2004067737A2 DK 2004000066 W DK2004000066 W DK 2004000066W WO 2004067737 A2 WO2004067737 A2 WO 2004067737A2
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Prior art keywords
atom
subtilase
seq
positions
variant
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PCT/DK2004/000066
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French (fr)
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WO2004067737A3 (en
Inventor
Allan Svendsen
Henriette Draborg
Nikolaj Tindbaek
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Novozymes A/S
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Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to EP04706617A priority Critical patent/EP1590454A2/en
Priority to US10/786,850 priority patent/US7294499B2/en
Publication of WO2004067737A2 publication Critical patent/WO2004067737A2/en
Publication of WO2004067737A3 publication Critical patent/WO2004067737A3/en
Priority to US11/928,576 priority patent/US20090148893A1/en
Priority to US12/879,410 priority patent/US8084241B2/en
Priority to US13/304,880 priority patent/US8383387B2/en
Priority to US13/743,839 priority patent/US8546122B2/en
Priority to US14/013,231 priority patent/US9169475B2/en
Priority to US14/863,058 priority patent/US9493758B2/en
Priority to US15/280,387 priority patent/US10144922B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to variants of TY145 subtilases and BPN' subtilases and to methods of construction such variants with altered properties, such as stability (e.g. thermo- stability or storage stability), Ca 2+ dependency, pH dependent activity.
  • stability e.g. thermo- stability or storage stability
  • Ca 2+ dependency e.g. Ca 2+ dependency
  • pH dependent activity e.g. thermo- stability or storage stability
  • Enzymes have been used within the detergent industry as part of washing formulations for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellulases, hemicellu- lases or mixtures of enzymes are also often used.
  • proteases with altered properties such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • the search for proteases with altered properties include both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g! genetic manipulation of the nucleic acid sequence encoding said proteases. Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific characteristic of the protein.
  • subtilases One family of proteases, which are often used in detergents, are the subtilases. This family has previously been further grouped into 6 different sub-groups by Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523. One of these sub-groups is the Subtilisin family which includes subtilases such as BPN', subtilisin 309 (SAVINASE ® , NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE ® NOVOZYMES A/S), subtilisin S41 (a subtilase from the psychrophilic Antarctic Bacillus TA41 , Davail S et al. 1994, The Journal of Biological Chemistry, 269(26), 99.
  • subtilases such as BPN', subtilisin 309 (SAVINASE ® , NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE ® NOVOZYMES A/S), subtilisin S41 (a subtilase from the psych
  • subtilisin S39 a subtilase from the psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11), pp. 1271-1279
  • TY145 a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577.
  • modelling of the three-dimensional structure of one subtilase on the basis of the three-dimensional structure of another subtilase may result in an incorrect three- dimensional structure because of structural differences.
  • the inventors of the present invention have elucidated the three-dimensional structure of the TY 45 subtilase and found that there are several differences between this and the three- dimensional structure of BPN' also belonging to the Subtilisin subgroup of subtilases.
  • This surprising difference in structure makes it advantageous to use the TY145 structure as basis for homology modelling of TY145 like subtilisins, which, in turn, will improve the ability to obtain desired changes in functionality by protein engineering.
  • Mi- yazaki K et al. 2000, J Mol Biol, 297, pp.1015-1026 discloses enhancement of the thermostability and activity of the psychrophilic protease subtilisin S41 by methods of directed evolution.
  • Wintrode TL et al. 2000, Journal of Biological Chemistry, 275 (41), pp.31635-31640 discloses conversion of a mesophilic subtilisin-like protease from Bacillus sphaericus SSI I into its psy- chrophilic counterpart by methods of directed evolution.
  • Wintrode et al. constructed the three-dimensional structural model of the SSII subtilase on basis of its homology with subtilisins Carlsberg, Savinase, BPN' and Thermitase.
  • the SSII subtilase pertain to the new group of TY145 like subtilases and thus the modelling of SSII based on the 3D structure of the BPN' like subtilases will likely give an inaccurate result.
  • the inventors have modified the amino acid sequence of a subtilase to obtain variants with improved properties, based on the three-dimensional structure of the subtilases TY145 and BPN'.
  • the variants have altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • the object of the present invention is to provide a method for constructing subtilases having altered properties, in particular to provide a method for constructing subtilases having altered properties as described above.
  • the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: i) analyzing the three-dimensional structure of the subtilase to identify, on the basis of an evaluation of structural considerations, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; ii) constructing a variant of the subtilase, which as compared to the parent subtilase, has been modified in the amino acid residue or structural part identified in i) so as to alter said property; and iii) testing the resulting subtilase variant for said property.
  • modification of the parent subtilase in certain regions and/or positions is expected to confer a particular effect to the thus produced subtilase variant
  • modification of the parent subtilase in any of such regions may also give rise to any other of the above-mentioned effects.
  • any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased peptidase activity.
  • APPENDIX 1 shows the structural coordinates for the solved crystal 3D structure of the TY145 subtilase.
  • Figure 1 shows a multiple alignment of 3D sequences of subtilases from TY145, TA39,
  • Figure 2 shows an alignment between the amino acid sequences of subtilisin BPN' and Savinase in order to define the BPN' numbering of Savinase.
  • Figure 3 shows a superposition of TY145 subtilase (light) and BPN' structures (dark), with spheres indicating ion-binding sites.
  • the TY145 ion-binding sites are light and the BPN' ion- binding sites are dark.
  • Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue. Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-S1 ("true" subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those de- scribed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • parent is in the context of the present invention to be understood as a protein, which is modified to create a protein variant.
  • the parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means.
  • the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide.
  • parent subtilase refers to a subtilase which is modified to create a subtilase variant.
  • variant is in the context of the present invention to be understood as a protein which has been modified as compared to a parent protein at one or more amino acid residues.
  • modification(s) or “modified” is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein.
  • the modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
  • modified protein e.g. "modified subtilase”
  • subtilase is to be understood as a protein which contains modification(s) compared to a parent protein, e.g. subtilase.
  • subtilase or "(a) TY145 like subtilase” should in the context of the pre- sent invention be understood as a subtilase belonging to the Subtilisin group according to Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 63% homology to TY145, SEQ ID NO:1. In the context of the present invention a TY145 subtilase has three ion-binding sites.
  • subtilase or "(a) BPN' like subtilase” should in the context of the present invention be understood as a subtilase belonging to the Subtilisin group according Siezen et al. Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 61% homology to BPN' SEQ ID NO:5.
  • Such a BPN' like subtilase is for example Savinase.
  • a BPN 1 subtilase has two, three or five ion-binding sites.
  • a BPN' like subtilase may, in the context of the present invention, belong to branch l-S of the subtilisins i.e. to branch I-S1, the "true” subtilisins or I-S2, the highly alkaline proteases (Siezen et al., Protein Engng. 4 (1991) 719-737).
  • Identities can be extracted from the same calculation.
  • the output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" and the "Similarity” between the two sequences.
  • the numbers calculated using UWGCG package version 9.1 is slightly different from the version 8.
  • position is in the context of the present invention to be understood as the number of an amino acid in a peptide or polypeptide when counting from the N-terminal end of said peptide/polypeptide.
  • position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to.
  • subtilases belonging to the TY145 subgroup i.e. subtilases obtained from TY145, TA39, TA41 and Bacillus sphaericus are numbered individually according to each of SEQ ID NO:1 ,2,3 and 4.
  • subtilases belonging to the TY145 subgroup are numbered individually according to their own sequence. However in order to determine homologous positions in such other subtilases an alignment with each of SEQ ID's NO:1 ,2,3 and 4 is conducted according to the GAP procedure described above. Subsequently the homologous positions are determined with reference to the most homologous of SEQ ID's NO: 1 ,2,3 and 4. Alternatively subtilases belonging to the TY145 subgroup can be numbered by reference to the positions of TY145 subtilase (SEQ ID NO:1).
  • Subtilases belonging to the BPN' subgroup refers to the positions of Subtilisin Novo (BPN') from B. amyloliquefaciens (SEQ ID NO:5).
  • BPMES Bacillus pumilus
  • the inventors of the present invention find that the subgroup of TY145 subtilases are different from BPN' subtilases based on the 3D structure comparison of the enclosed 3D structure of TY145 and the BPN' 3D structure but also indicated from the sequence homology between TY145 and BPN'.
  • a TY145 subtilase is in the context of the present invention to be under- stood as a subtilase which has at least 63% homology to SEQ ID NO:1.
  • said TY145 subtilase may have at least 65%, such as at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to TY145, i.e. to SEQ ID NO:1.
  • a TY145 subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of TY145, i.e. it may be homologous to the three-dimensional structure defined by the structure coordinates in Appendix 1.
  • a set of structure coordinates for a protein or a portion thereof is a relative set of points that define a shape in three dimensions, it is possible that an entirely different set of coordinates could define an identical or a similar shape.
  • slight variations in the individual coordinates may have little or no effect on the overall shape.
  • variations in coordinates may be generated because of mathematical manipulations of the structure coordinates.
  • the structure coordinates of Appendix 1 (TY145 structure) may be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • said variations may be due to differences in the primary amino acid sequence. If such variations are within an acceptable standard error as compared to the structure coor- dinates of Appendix 1 said three-dimensional structure is within the context of the present invention to be understood as being homologous to the structure of Appendix 1.
  • the standard error may typically be measured as the root mean square deviation of e.g.
  • RMS root mean square deviation
  • similarities in 3D structure of different subtilases can be described by the content of homologous structural elements, and/or the similarity in amino acid or DNA sequence. For sequences having no deletions or insertions a RMS for the calcium atoms can be calculated.
  • TY145 like subtilases examples include the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41, herein also called TA41 subtilase (Davail S et al., 1994, J.
  • subtilisin protease S39 derived from the Antarctic Bacillus TA39, herein also called TA39 subtilase (Narinx E et al., 1997, Protein Engineering, 10 (11), 1271-1279). Recently a three-dimensional structure of a subtilisin homologous with the TY145 subtilisins was published in the Protein Data Bank (Accession No:1EA7).
  • a preferred embodiment of the present invention is a parent subtilase or a subtilase variant which is at least 63% homologous to the sequence of SEQ ID NO:1 , preferably at least 65%, at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence of SEQ ID NO:1 , and optionally said subtilase further comprises the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites and wherein the Strong ion-binding site of the BPN' like subtilases is not present, and with the exception of the TY145 subtilase, the TA39 subtilase, the TA41 subtilase, and the Bacillus s
  • the TY145 subtilase of the present invention is encoded by an isolated nucleic acid sequence, which nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:20.
  • isolated nucleic acid sequence encoding a TY145 subtilase of the invention hy- bridizes with a complementary strand of the nucleic acid sequence shown in SEQ ID NO:20 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
  • Suitable experimental conditions for determining hybridization at *low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves pre- soaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
  • the filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least * 55°C (low stringency), more preferably at least 60°C (medium stringency), still more preferably at least 65°C (medium/high stringency), even more preferably at least 70°C (high stringency), and even more preferably at least 75°C (very high stringency).
  • a BPN' subtilase is in the context of the present invention to be understood as a subtilase which has at least 61% homology to SEQ ID NO:5.
  • said BPN' subtilase may have at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to BPN', i.e. to SEQ ID NO:5.
  • a BPN' subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of BPN'- as defined by the structure coordinates given, in PDB Nos. 1SBT and 1GNS (Protein Data Bank), or one of the several other structures of BPN' that are accessible from the Protein Data Bank. Variations between homologous structures may occur for several reasons as described above.
  • a BPN' subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the BPN' subti- lases as described above, and in addition such subtilases does preferably not have further structural characteristics which are not present in the BPN 1 subtilases as described herein.
  • a BPN' subtilase of the present invention may have the necessary percentage of similarity with SEQ ID NO:5.
  • BPN' like subtilases examples include the subtilisin 309 (PDB NO:1SVN SAVINASE ® , NOVOZYMES A/S) and subtilisin Carlsberg (ALCALASE ® , NOVOZYMES A/S), among others.
  • subtilase consists of 6-8 helices, 11 strands of which 7 are central in a twisted beta-sheet. Two ion-binding sites are mentioned, one of which is the so called "Weak" calcium-binding site. It was later discovered that for some structures (subtilisin DY PDB no. 1 BH6, 1998), this calcium-binding site was shown to be a Na (sodium) binding site when the calcium concentration in the crystallization medium was low. Thus, in the following we refer to ion-binding sites instead of calcium-binding sites.
  • the BPN' subtilase of the present invention is encoded by an isolated nucleic acid sequence, which nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:21.
  • isolated nucleic acid sequence encoding a BPN' subtilase of the invention hybridizes with a complementary strand of the nucleic acid sequence shown in SEQ ID NO:21 preferably under low stringency conditions, but at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
  • the TY145 subtilase was used to elucidate the three-dimensional structure forming the basis for the present invention.
  • the structure of TY145 was solved in accordance with the principle for x-ray crystallographic methods, for example, as given in X-Ray Structure Determination, Stout, G.K. and Jensen, L.H., John Wiley & Sons, Inc. NY, 1989.
  • the structural coordinates for the solved crystal structure of TY145 are given in standard PDB format (Protein Data Bank, Brookhaven National Laboratory, Brookhaven, CT) as set forth in Appendix 1. It is to be understood that Appendix 1 forms part of the present application.
  • CA refers to c-alpha (carbon atoms) or to calcium ions, (however to avoid misunderstandings we use the full names "c-alpha atoms” and “calcium” or “ion” in the present specification).
  • Amino acid residues are given in their standard three-letter code.
  • the attached structural coordinates contain the protease structure, and an inhibitor structure CI2 as well as water molecules.
  • the protease coordinates has a chain identification called A, whereas the CI2 inhibitor is called B, the calcium ions are called C, and the water is W.
  • the positions of the men- tioned residues refer to the sequence of TY145 as disclosed in SEQ ID NO:1.
  • the structure of TY145 shows the same "overall" fold as found in the S8 family of subtilisins.
  • the structure comprises a twisted beta-sheet with 7 strands arranged in the following sequential order S2, S3, S1 , S4, S5, S6, S7.
  • the TY145 like subtilases are shown to lack the well-known Strong ion-binding site of the BPN' subtilases.
  • the TY145 subtilases have two ion-binding sites which are not present in the BPN' subtilisin structures. This can be seen in the structural alignment presented in Figure 3. These additional ion-binding sites are hereinafter referred to as "Near” and “Far” according to their distance to the Weak ion-binding site.
  • the ion-binding sites of TY145 are located at: Weak - calcium atom named C 314, Near - calcium atom named C 312, and Far - calcium atom named C 313 in the PDB table (Appendix 1).
  • the position of an ion-binding site can be defined by the distance to four specific atoms in the core structure.
  • the distance from the ion-binding site to the c-alpha atoms of the three active site residues has been chosen.
  • the residues Ser, His and Asp in the active site are highly conserved. In TY145 they are Asp35, His72 and Ser251.
  • the fourth distance chosen is the distance to the c-alpha atom of the amino acid residue coming first after the active site serine residue in the sequence (herein after called "next to Ser"); in the 3D structure of TY145 it is Met252. • .
  • the distance between: a) the Weak ion-binding site and i) Asp c-alpha atom is 17.50-19.50A, ii) His c-alpha atom is 21-23A, iii) Ser c-alpha atom is 13.80-15.80A, iv) next to Ser c-alpha atom is 15.80-
  • Ser c-alpha atom is 20-22A, iv) next to Ser c-alpha atom is 19.50-21.50A, c) the Near ion-binding site and i) Asp c-alpha atom is 27-29A, ii) His c-alpha atom is
  • the distances may vary from one subtilase to the other, and as described above, the Weak ion binding site may also bind to a sodium ion.
  • the present distances are given with a calcium ion in the structure. If a sodium ion was bound instead the distances would be shifted a little bit.
  • the distances can vary ⁇ O. ⁇ A, preferably +0.7A, ⁇ 0.6A, ⁇ O. ⁇ A, ⁇ 0.4A, or most preferably ⁇ 0.3A.
  • the peptide structure circumscribing the Weak ion- binding site is composed of the amino acid residues placed in positions 182-189 and 221- 227 with the coordinating atoms being the backbone carbonyl oxygen atom of residues G182, A187, L184 and two water molecules.
  • the peptide structure circumscribing the Near ion-binding site is composed of residues 212- 225 with the coordinating atoms being the backbone carbonyl oxygen atom of residues I220 and T215, the oxygens from the carboxylic acids of residues D225 and D218 and the amid group of residue Q222.
  • the peptide structure circumscribing the Far ion-binding site is composed of residues 288- 306 with the coordinating atoms being the backbone carbonyl oxygen atom of residues G298, G296 and I289, the oxygens from the carboxylic acids of residues D300 and D288, and two water molecules.
  • the structure of the TY145 like subtilase can be divided into a "common subtilase-like" region, an "intermediate” region and a "non- homolo-gous" region.
  • the active site can be found in the common subtilase-like region, which is structurally closely related to the BPN' structures.
  • the common subtilase-like region is composed of residues 88-128 and 225-284, and contains the alpha-helix H3 and the central alpha-helix H5 in which the active site serine residue is situated in the N-terminal part.
  • the common subtilase-like region has an RMS lower than 1.2.
  • the structure of the TY145 like subtilase differs from the BPN' structures to a greater extent.
  • the intermediate region consist of residues 24-45, 48-58, 65-66, 67-85, 134-174, 175-196, 202-212 and 287-290.
  • the intermediate region has an RMS higher than 1.2 and lower than 1.8.
  • the relationships between the three-dimensional structure and functionality are potentially difficult to predict in this region of the TY145 like subtilases.
  • the nonhomologous region consists of residues 5-15, 16-23, 86-87, 129-133, 197-201 , 213-
  • the nonhomologous region has a RMS higher than 1.5, which also pertains to residues 65-66 from the intermediate region.
  • the group comprising residues 5-15 and 299-311 has an RMS between 2.1-2.2. The relationships between the three-dimensional structure and functionality are very difficult to predict in this region of the TY145 like subtilases.
  • the regions in areas A1-T5, N16-T24, A46-Q51 , S58-C66, G84-G90, S129-K134, S129- K134, S173-S175, V196-T201 , N212-R224, A284-V286, K290-D299 and V310-K311 in the TY145 structure differs significantly from the other S8 family subtilisins (including the BPN 1 type subtilisins) in c-alpha atom coordinates. An RMS cannot be calculated for these last residues as there are no homologous c-alpha atoms in the compared subtilases.
  • a model structure of a TY145 like subtilase or a BPN' like subtilase can be built using the Homology program or a comparable program, e.g., Modeller (both from Molecular Simulations, Inc., San Diego, CA).
  • the principle is to align the amino acid sequence of a protein for which the 3D structure is known with the amino acid sequence of a protein for which a model 3D structure has to be constructed.
  • the structurally conserved regions can then be built on the basis of consensus sequences.
  • loop structures can be inserted, or sequences can be deleted with subsequent bonding of the necessary residues using, e.g., the program Homology. Subsequent relaxing and optimization of the structure should be done using either Homology. or another molecular simulation, program," e.g., CHARMm from Molecular Simulations.
  • the present invention comprises a method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 subtilase on the three-dimensional structure of a TY145 subtilase to produce a three-dimensional structure of the parent TY145 subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent TY145 subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions
  • subtilase TY145 subtilase; f) isolating the produced subtilase; g) purifying the isolated subtilase and h) recovering the purified subtilase.
  • the present invention comprises a method of producing a variant of a parent Subtil- isin family subtilase, such as a BPN' like subtilase, the variant having at least one altered property as compared to the parent Subtilisin family subtilase, the method comprising: a) modelling the parent Subtilisin family subtilase on the three-dimensional structure of a Subtilisin family subtilase to produce a three-dimensional structure of the parent Subtilisin family subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 like subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of , the parent Subtilisin family subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent Subtilisin family subtilase to produce a nucleic acid sequence en
  • the present invention comprises a method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 like subtilase on the three-dimensional structure of a TY145 like subtilase to produce a three-dimensional structure of the parent TY145 like subti- lase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a Subtilisin family subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 like subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent TY145 like subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino ac- ids at a position corresponding to said
  • the TY145 subtilases has two new ion-binding sites not present in the BPN' subtilisin structures but lacks the Strong ion-binding site of the BPN' subtilases. Stability of the ion-binding site is of crucial importance for the functionality of the enzyme. Therefore alterations of the amino acid residues close to the ion-binding sites are likely to result in alterations of the stability of the enzyme.
  • the positions which may be modified are located: Weak: at a distance of 10A or less around calcium atom named C 314, Near: at a distance of 10A or less around calcium atom named C 312, and Far: at a distance of 10A or less around calcium atom named C 313 in the PDB table (Appendix 1).
  • Stabilisation of the ion-binding sites of TY145 may possibly be obtained by alterations in the positions close to the sites. Positions located at a distance of 10A or less to the ion-binding sites of TY145 (SEQ ID NO:1) are:
  • variants with improved calcium stability can be constructed.
  • Variants with alterations close to the Near ion-binding site are I220S,T and T215S
  • variants with alterations close to the Far ion-binding site are G298A,S,T and G296A,S,T
  • variants with alterations close to the Weak ion-binding site are V185T and I221N,D,T.
  • TY145 with extra ion-binding site The Strong ion-binding site from the BPN' subtilases can be transplanted into TY145 (or other subtilases in TY145 subgroup) by deletion(s) of or in the region H83-G90 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues.
  • a preferred variant has the whole region deleted and a subsequent insertion between A82 and V91 of the sequence LNNSIG.
  • the Far and Near ion-binding sites in TY145 can be removed with guidance from the three-dimensional structure of BPN' and Sav-. inase (or others in BPN 1 group).
  • Removal of the Far site can be done by deletion(s) of or in the region K290-D300 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues.
  • a preferred variant has the whole region deleted and a subsequent insertion between I289 and Y301 of the sequence GDS or DST.
  • the substitution S303Y is further added.
  • Removal of the Near site can be done by deletion(s) of or in the region N212-R224 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues.
  • a preferred variant has the whole region deleted and a subsequent insertion of a proline or alanine residue between G211 and D225.
  • the Strong ion-binding site in BPN' like subtilases can be removed. Exemplified in Savinase, the removal can be done by deletion of or in the region L75-G80 (BPN' numbering) and subsequent insertion of one or more amino acid residues.
  • a preferred variant has the whole re- gion deleted and a subsequent insertion of residues 84-88 from TY145.
  • substitutions L82Y and Q2A,N can be applied. Alteration of thermostability
  • a variant with improved stability may be obtained by substitution with proline, introduction of a disulfide bond, altering a hydrogen bond contact, altering charge distribution, introduction of a salt bridge, filling in an internal structural cavity with one or more amino acids with bulkier side groups (in e.g. regions which are structurally mobile), substitution of histidine residues with other amino acids, removal of a deamidation sites, or by helix capping.
  • Regions with increased mobility The following regions of TY145 have an increased mobility in the crystal structure of the enzyme, and it is presently believed that these regions can be responsible for stability or activity of TY145. Especially thermostabilisation may possibly be obtained by altering the highly mobile regions. Improvements of the enzyme can be obtained by mutation in the below regions and positions. Introducing e.g. larger residues or residues having more atoms in the side chain could increase the stability, or, e.g., introduction of residues having fewer atoms in the side chain could be important for the mobility and thus the activity profile of the enzyme, The regions can be found by analysing the B-factors taken from the coordinate file in Appendix 1 , and/or from molecular dynamics calculations of the isotropic fluctuations. These can be obtained by using the program CHARMm from MSI (Molecular Simulations Inc.).
  • 141-146 i.e. 141 , 142, 143, 144, 145, 146)
  • 150-152 i.e.150, 151 , 152
  • 169-171 i.e. 169, 170, 171 200-201
  • 211-220 i.e. 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220
  • 242-243 268-270 i.e. 268, 269, 270.
  • 38-50 i.e. 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50
  • 57-69 i.e. 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69
  • 84-92 i.e. 84, 85, 86, 87, 88, 89, 90, 91, 92
  • 107-110 i.e. 107, 108, 109, 110
  • 239-243 i.e. 239, 240, 241 , 242, 243
  • 265-266 Preferably the regions 57-69 and 84-92.
  • a TY145 variant of the present invention with improved stability, e.g. thermostability, as compared to the parent TY145 may be obtained by introducing new inter-domain or intra-domain bonds, such as by establishing inter- or intra-domain disulfide bridges.
  • a further aspect of the present invention relates to a method for producing a variant of a parent TY145 comprising the methods described in the paragraph "Methods of preparing TY145 like or BPN' like subtilase variants" herein.
  • amino acid residues iden ⁇ tified in the amino acid sequence of SEQ ID NO:1 are contemplated as being suitable for cysteine replacement.
  • disulfide bridges may possibly form in a variant of TY145.
  • the substitutions are: G26C+A95C; A167C+T254C; R203C+G292C and V228C+A284C:
  • Similar residues suitable for cysteine replacement in homologous subtilases such as TA39, TA41 can be elucidated by finding the homologous positions in the alignment of Figure 1.
  • Concerning another TY145 like sequence the homologous positions suitable for cysteine replacement can be selected by aligning said TY145 like sequence with all of the sequences of Figure 1 using the GAP analysis method as described above.
  • the suitable residues can then be selected in accordance with the homologous positions in the most homologous of SEQ ID's NO:1 ,2,3 and 4 which are the sequences of the subtilases aligned in Figure 1.
  • a variant with improved stability (typically improved thermostability) as compared to the parent subtilase may be obtained by changing the surface charge distribution of the subtilase. For example, when the pH is lowered to about 5 or below histidine residues typically become positively charged and, consequently, unfavorable electrostatic interactions on the protein surface may occur. By engineering the surface charge of the subtilase one may avoid such unfavorable electrostatic interactions that in turn lead to a higher stability of the subtilase.
  • a further aspect of the present invention relates to a method for constructing a variant of a parent subtilase, the method comprising: a) identifying, on the surface of the parent subtilase, preferably a TY145 like or a BPN' like subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one amino acid residue se- lected from the group consisting of Asp, Glu, Arg, Lys and His with an uncharged amino acid residue; c) optionally repeating steps a) and b) recursively; d) optionally, making alterations each of which is an insertion, a deletion or a substitution of an amino acid residue at one or more positions other than b); e) preparing the variant resulting from steps a) - d); f) testing the stability of said variant; and g) optionally repeating steps a
  • the above method may be employed. Again, compared to the above method, the only difference being steps a) and b) which, in this case, read: a) identifying, on the surface of the parent subtilase, at least one charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one charged amino acid resi- due identified in step a) with an amino acid residue having an opposite charge.
  • Asp may be substituted with Arg, Lys or His;
  • Glu may be substituted with Arg, Lys or
  • thermostability of a subtilase can be obtained by subjecting the subtilase in question to analysis for secondary structure, identifying residues in the subtilase having dihedral angles ⁇ (phi) and ⁇ (psi) confined to the intervals [-90° ⁇ -40° and -180° ⁇ 180°], preferably the intervals [-90° ⁇ -40° and 120° ⁇ 180°] or [-90° ⁇ -40° and -50° ⁇ 10°] and excluding residues located in regions in which the subtilase is characterized by possessing ⁇ -helical or ⁇ -sheet structure.
  • proline residues are generally not compatible with ⁇ -helical and ⁇ -sheet secondary conformations. If a proline residue is not already at the identified position(s), the naturally occurring amino acid residue is substituted with a proline residue, preferably by site directed mutagenesis applied on a gene encoding the subtilase in question.
  • TY145 like subtilases proline residues can be introduced at positions 18, 115, 185, 269 and 293. Accordingly, a preferred TY145 variant has one or more of the substitu- tions: Q18P, D115P, V185P, T269P and I293P.
  • TY145 a mesophilic-derived enzyme obtained from crystal structure
  • TA41 a psychrophilic derived enzyme obtained from modelling
  • the higher mobility regions in TA41 compared to TY145 measured by molecular dynamics simulation indicates important regions for the low temperature activity, of the enzyme TA41 which can be transferred to TY145.
  • the regions in TA41 are: 16-22 (i.e. 16, 17, 18, 19, 20, 21, 22),
  • 40-73 i.e. 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59,
  • 118-131 i.e.118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131
  • 140-161 i.e.140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161
  • 140-161 i.e.140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161
  • Regions closest to the active site and the substrate binding site are regarded as preferred in relation to making higher activity at low temperature for TY145: 40-73, and 140-161 , preferably 65-73 and 140-150.
  • the regions in TY145 should be modified to be more mobile for example by substitution with small less rigid residues, i.e. residues with smaller side chains (such as Gly, Ala, Ser, Thr or Val), into the TY145 backbone.
  • the other regions in TA41 are most interesting for stabilisation of the psychrophilic enzyme. These regions can easily be found in TA39 as well or in other homologous enzymes, also non psychrophilics.
  • the regions around the active site and the substrate binding site are the regions most likely involved in the low temperature functionality. Below are suggestions for transferring the low temperature activity of TA41 and homologous sequences to TY145-like sequences and the BPN'-like sequences:
  • Savinase is numbered according to BPN'.
  • Preferred Savinase variants are V28l, I35V, T71S, I72A, A73L, M175V and T224S.
  • Examples of core variants of TY145 are: V31 I, V80A, T79S.
  • the alterations of the TY145-like sequences and the BPN'-like sequences can be single mutations or combinations of the suggested mutations.
  • the substrate binding site is identified by the residues in contact with a substrate model, such as the CI2 inhibitor.
  • a substrate model such as the CI2 inhibitor.
  • the 3D structure coordinates of the TY145 subtilase with CI2 bound in the active site can be found in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favor- able interactions found within a sphere 10 A from the substrate molecule, in particular within a sphere of 6 A from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions.
  • the following residues of the TY145 subtilase (SEQ ID NO:1), are within a distance of 6A from the CI2 inhibitor and thus believed to be involved in interactions with said substrate: 35, 36, 70, 72, 106, 109, 110, 111 , 112, .113, 114, 117, 139, 140, 141 , 142, 143, 144, 145, 147, 150, 167, 168, 169, 170, 171 , 172, 173, 174, 177, 180, 207, 239, 247, 148, 149, 150, 151 and 252.
  • the invention consequently further relates to a subtilase, in which either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent RP-II protease is/are deleted or substituted with a residue of a different amino acid.
  • the Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
  • Asn-Gly sequences can be found in the following positions: B. sphaericus: 198-199, 240-241 TY145: 87-88, 109-110, 199-200 TA41 : 83-84, 198-199 TA39: 88-89, 198-199
  • the present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
  • Tyrosines can be found in the following positions:
  • TY145 15, 39, 92, 103, 113, 156, 158, 202; 219, 240, 244, 287, 301 , 307, TA41 : 15, 91, 102, 112, 155, 157, 179, 201 , 218, 235, 243,
  • the present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
  • Methionines can be found in the following positions: B. sphaericus: 138, 251 ,
  • the present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
  • the present invention also encompasses any of the above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof.
  • Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged.
  • Such combinations comprise the positions: 222 (improves oxidation stability), 218 (improves thermal stability), substitutions in the Ca 2+ -binding sites stabilizing the enzyme, e.g. position 76, and many other apparent from the prior art (all positions according to BPN' numbering).
  • subtilase variant described herein may advantageously be com- bined with one or more modification(s) in any of the positions:
  • N76D+V104A or other combinations of the modifications K27R, N76D, S101G, S103A, V104N, V104Y, V104I, V104A, N123S, G159D, A232V, Q236H, Q245R, N248D, N252K,
  • T274A in combination with any one or more of the modification(s) mentioned above exhibit improved properties.
  • a particular interesting variant is a variant, which, in addition to modifications according to the invention, contains the following substitutions: S101 G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
  • subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, 131 and 194, preferably as 129K, 131 H and 194P modifications, and most preferably as P129K, P131 H and A194P modifications. Any of those modification(s) are expected to provide a higher expression level of the subtilase variant in the production thereof.
  • the subtilase variants i.e. the TY145 and BPN' variants of the present invention may be produced by any known method within the art and the present invention also relates to nucleic acid encoding a subtilase variant of the present invention, a DNA construct comprising said nucleic acid and a host cell comprising said nucleic acid sequence.
  • natural occurring proteins may be produced by culturing the organism expressing the protein and subsequently purifying the protein or it may be produced by cloning a nucleic acid, e.g. genomic DNA or cDNA, encoding the protein into an expression vector, introducing said expression vector into a host cell, culturing the host cell and purifying the expressed protein.
  • protein variants may be produced by site-directed mutagenesis of a parent protein, introduction into expression vector, host cell etc.
  • the parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof.
  • a parent subtilase, or subtilase or subtilase variant of the invention may be produced by site-directed mutagenesis of a parent protein, introduction into expression vector, host cell etc.
  • the parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof.
  • mutations random and/or site directed
  • suitable techniques reference is made to Molecular cloning: A laboratory manual (Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F.
  • variants could be constructed by:
  • Random Mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.
  • the random mutagenesis of a DNA sequence encoding a parent subtilase may be conven- iently performed by use of any method known in the art.
  • a further aspect of the present invention relates to a method for generating a variant of a parent subtilase, wherein the variant exhibits an altered property, such as increased thermostability, increased stability at low pH and at low calcium concentration, relative to the parent subtilase, the method comprising:
  • step (b) expressing the mutated DNA sequence obtained in step (a) in a host cell
  • Step (c) screening for host cells expressing a subtilase variant which has an altered property relative to the parent subtilase.
  • Step (a) of the above method of the invention is preferably performed using doped primers.
  • the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis.
  • the random mutagenesis may be performed by use of any combination of these mutagenizing agents.
  • the mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS) ; sodium bisulphite, formic acid, and nucleotide analogues.
  • UV ultraviolet
  • mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties.
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions that are to be changed.
  • the doping or spiking may be done so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the subtilase enzyme by any published tech- nique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
  • the doping is carried out using "constant random doping", in which the percentage of wild-type and modification in each position is predefined.
  • the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues.
  • the doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% modifications in each position.
  • An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
  • the doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided (L.J. Jensen et al. Nucleic Acid Research, 26, 697-702 (1998).
  • PCR-generated mutagenesis When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent subtilase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, 1 , 1989, pp. 11-15). A mutator strain of £. coli (Fowler et al., Molec. Gen. Genet, 133, 1974, 179-191), S.
  • cere- viseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the subtilase by, e.g., transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain.
  • the mutated plasmid may be subsequently transformed into the ex- pression organism.
  • the DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent subtilase.
  • the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenising agent.
  • the DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harbored in the cell.
  • the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
  • the mutated DNA sequence may be conveniently amplify prior to perform- ing the expression step b) or the screening step c). Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR- generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme. Subsequent to the incubation with or exposure to the mutagenising agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place.
  • the host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment.
  • suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermo- philus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circu- lans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gram negative bacteria such as E. coli.
  • the mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.
  • the random mutagenesis may be advantageously localised to a part of the parent subtilase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
  • the localised or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art.
  • the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
  • the random mutagenesis may be carried out by the following steps: 1. Select regions of interest for modification in the parent enzyme
  • Suitable dope algorithms for use in step 6 are well known in the art.
  • One such algorithm is described by Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11 :29- 38.
  • Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A, and Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).
  • a recombinant expression vector comprising a nucleic acid sequence encoding a subtilase variant of the invention may be any vector that may conveniently be subjected to recombinant DNA procedures and which may bring about the expression of the nucleic acid se- quence.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • a suitable vector include a linear or closed circular plasmid or a virus.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAM ⁇ l
  • Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1.
  • the origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehr- lich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome, in particular it may contain nucleic acid, sequences facilitating integration into the genome by homologous or non-homologous recombination.
  • the vector system may be a single vector, e.g. plasmid or virus, or two. or more vectors; e.g. plasmids or virus', which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the vector may in particular be an expression vector in which the DNA sequence encoding the subtilase variant of the invention is operably linked to additional segments or control sequences required for transcription of the DNA.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence encoding the subtilase variant.
  • Additional segments or control sequences include a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum the control sequences include a promoter and transcriptional and translational stop signals.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for use in bacterial host cells include the promoter of the Ba- cillus subtilis levansucrase gene (sacB), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amylolique- faciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacil- lus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-
  • phage Lambda P R or P L promoters examples include the phage Lambda P R or P L promoters or the E. coli lac, trp or tac promoters or the Streptomyces coelicolor agarase gene (dagA). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.
  • promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable al- pha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusahum oxysporum trypsin-like protease (as described in U.S.
  • Patent No. 4,288,627 which is incorporated herein by reference
  • Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.
  • Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpiA promoter.
  • promoters for use in yeast host cells include promoters from yeast gly- colytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
  • yeast host cells are described by Romanos et al., 1992, Yeast 8:423- 488.
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • SV40 Simian Virus 40
  • RSV Rous sarcoma virus
  • BPV bovine papilloma virus
  • Suitable promoters for use in mammalian cells are the SV40 promoter (Subra- mani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • the DNA sequence encoding a subtilase variant of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like am- picillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs.
  • bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance.
  • a frequently used mammalian marker is the dihydrofolate reductase gene (DHFR).
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyl- transferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate.
  • sC sulfate adenyltransferase
  • trpC anthranilate synthase
  • glufosinate resistance markers, as- well as equivalents from other species.
  • amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.
  • selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the enzyme.
  • the secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
  • More than one copy of a nucleic acid sequence encoding an enzyme of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence.
  • Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
  • the nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention.
  • an activator e.g., a trans-acting factor
  • a chaperone e.g., a chaperone
  • processing protease e.g., a factor that is functional in the host cell of choice.
  • the nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.
  • the DNA sequence encoding a subtilase variant of the present invention may be either homologous or heterologous to the host cell into which it is introduced. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to an- other promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment.
  • the term "homologous” is intended to include a DNA sequence encoding an enzyme native to the host organism in question.
  • heterologous is intended to include: a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic. sequence.
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present subtilase variants, such as prokaryotes, e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.
  • prokaryotes e.g. bacteria or eukaryotes
  • fungal cells e.g. yeasts or filamentous fungi
  • insect cells e.g. yeasts or filamentous fungi
  • plant cells or mammalian cells examples of bacterial host cells which, on cultivation, are capable of producing the subtilase variants of the invention are gram-positive bacteria such as strains of Bacillus, e.g. strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
  • amyloliquefaciens B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram-negative bacteria such as Escherichia coli or Pseudomonas sp.
  • the transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the enzyme When expressing the subtilase variant in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
  • the enzyme When expressing the subtilase variant in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or it may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
  • yeast cells include cells of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansehula, or Yarrowia.
  • the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansehula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231).
  • yeast Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F , Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980.
  • the biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J.; and Stopani, A.O.M.; editors; 2nd edition, 1987; The Yeasts,.
  • Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920.
  • filamentous fungal cells include filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra), in particular it may of the a cell of a species of Acremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusa um, such as F. bact di- oides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. hetero- sporum, F.
  • a species of Acremonium such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. ory
  • insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions may suitably be as described in WO 89/01029 or WO 89/01028.Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222; EP 397,485).
  • mammalian cells examples include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621 ; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341 ; Loyter et al., Proc. Natl. Acad. Sci.
  • Mammalian cells may be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
  • the above mentioned host cells transformed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
  • the medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Aca- demic Press, CA, 1991).
  • the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates.
  • the enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chroma- tographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the enzyme in question.
  • the enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activ- ity are known in the art.
  • the enzymes of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or ex- traction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or ex- traction
  • IEF isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • heterologous host cell When an expression vector comprising a DNA sequence encoding an enzyme of the present invention is transformed/tra ⁇ sfected into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme.
  • An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell.
  • homologous impurities mean any impurity (e.g. other polypeptides than the: enzyme of the invention) which originates from the h ⁇ . mologous cell where the enzyme. of the invention is originally obtained from.. . . .
  • the enzyme of the invention may be added to and thus become a component of a detergent composition.
  • the detergent composition of the invention may for example be formulated as a hand or ma- chine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
  • the invention provides a detergent additive comprising the enzyme of the invention.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • proteases include those of animal, vegetable or microbial origin. Micro- bial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
  • Preferred commercially available protease enzymes include Alcalase ® , Savinase ® , Primase ® , Duralase ® , Esperase ® , Ovozyme ® and Kannase ® (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM FN3TM and FN4TM (Genencor International Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1 ,372,034), P.
  • lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • Preferred commercially available lipase enzymes include Lipex ® , Lipolase ® and Lipolase Ul- tra ® (Novozymes A/S).
  • Amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, ⁇ - amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1 ,296,839.
  • Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
  • amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.). 5
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium ox- 0 ysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having colour care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as 5 those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • cellulases include Celluzyme ® and Carezyme ® (Novozymes A/S), ClazinaseTM, ; and Puradax HATM (Genencor International Inc.), and KAC-500(B)TM (Kao Corporation).
  • Peroxidases/Oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. 5 Commercially available peroxidases include Guardzyme ® (Novozymes A/S).
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e. a separate additive or a com- 0 bined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating mate- 5 rials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of .1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1% to 60% by weight.
  • the detergent will usually contain from about 1 % to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha
  • the detergent When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside; alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoetha- nolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides").
  • a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside; alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoetha- nolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • the detergent may contain 0-65 % of a detergent builder or complexing agent such as zeo- lite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethyl- enediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeo- lite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethyl- enediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 0 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hy- drotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hy- drotropes, tarnish inhibitors, or perfumes.
  • any enzyme in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liqour, preferably 0.05-50 mg of enzyme protein per liter of wash liqour, in particular 0.1-10 mg of enzyme protein per liter of wash liqour.
  • the enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
  • Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland. Especially type EMPA 116 (cotton textile stained with blood, milk and ink) and EMPA 117 (polyester/cotton textile stained with blood, milk and ink).
  • the present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
  • the expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well-known proce- dures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • a library based on Savinase positions V28, I35, T71 , I72, A73, M175 and T224 (BPN' numbering) have been synthesized.
  • the library contains exclusively TY145-suggested alterations and covers the introduced mutations V28I,A,L; I35V,A,L; T71S; I72A,G,V; A73L,G; M175V.A; T224S.A introduced in oligopeptides, some of which are doped.
  • Doping of nucleotide bases from a desired doping of individual amino acid residues, which is used for the example below, can be calculated as described at page 18 herein. In the attached sequence listing, the doped nucleotides below have been given the nucleo- tide symbols recommended by the WIPO Standard ST25.
  • the constructed oligopeptide primers are listed below.
  • the primers are named after which positions are subject to modifications, thus 28-35-CN may have alterations in positions 28 and 35, 71-72-73-NC may have alterations in positions 71 , 72 and 73, and so forth.
  • the Savinase gene is used as template for five individual PCR reactions under standard PCR conditions (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) where the oligos are combined as follows: 317 with 28-35-CN, 28-35-NC with 71-72-73-CN (a mixture of 80% (I) 71-72-73-CN, 10% (II) 71-72-73-CN and 10% (III) 71-72-73-CN), 71-72-73-NC with 175-CN (a mixture of 90% (l)175-CN and 10% (ll)175-CN), 175-NC with 224-CN, 224-NC with 139, giving PCR products of 125 bp, 126 bp, 3,12 bp, 165 bp and 158 bp respectively.
  • the library is assembled by an additional PCR reaction where the five PCR products are mixed in equal molar amounts. Thereby the library contains a large number of different Savinase variants altered in one or more of the mentioned positions.
  • the PCR reaction was assembled using a PTC-200 DNA Engine (MJ Research, Watertown, MA) and the following cycling parameters: 1 cycle of 2 min at 94 °C followed by 25 cycles of 30 sec at 94 °C, 30 sec at 55 °C and 1 min at 68 °C, and 1 cycle of 2 min at 68 °C.
  • the library was cloned by PCR multimerization (Shafikhani et al. 1997) into Savinase expression vector psx222 and transformed into a B. subtilis host for expression. Subsequently Savinase variants can be isolated from the library, purified and characterized.
  • properties from a BPN' like subtilase could be transferred to TY145 like subtilase by applying a similar procedure.
  • the Savinase regions (BPN' numbering) are deleted and the TY145 re- gions (SEQ ID NO:1) are inserted instead.
  • regions can be selected for transfer between the psycrophiles TA41/TA39 and BPN' type protease like Savinase, or from TA39/TA41 to TY145 type non-psychrophilic subtilases.
  • Savinase variants modified by receiving respectively segments II (Hybrid II), IV (Hybrid IV) or l+ll (Hybrid l+ll) from TY145 were observed to exhibit subtilase activity as determined by the formation of clearing zones on skim milk powder plates.
  • the below mentioned highly mobile regions in the TA39 subtilase S39 and the TA41 subti- lase S41 have been selected for transfer to Savinase.
  • the Savinase regions (BPN' numbering) are deleted and the S39 regions or S41 regions are inserted instead.
  • the S39 and S41 regions are numbered according to Figure 1.
  • regions can be selected for transfer between the psychrophiles TA41/TA39 and TY145 type non-psychrophilic subtilases.
  • Savinase variant V104S is used as acceptor for the S39 segment II.
  • Savinase variants modified by receiving respectively segments I or II from S39 were observed to have subtilase activity against the substrate suc-AAPF-pNA (Suc-Ala-Ala-Pro-Phe- pNA).
  • the subtilase activity was determined in a temperature profile assay where specific activities i.e. micromole substrate per minute per mg enzyme against before mentioned substrates, were determined at every 5 degrees Celsius. The measurements were done in a Tris-base buffer pH 9.
  • subtilase activity in suc-AAPF-pNA 100 uL 1.56 mM Suc-Ala-Ala-Pro-Phe-pNA in 0.1 M Tris was added to 100 uL Tris-base, pH 9.0 buffer and 20 uL enzyme.
  • the devel- opment of the degradation product pNA paranitrophenol was measured as initial velocities at 405 nm on an Elisa Reader for 1 minute.
  • the Savinase variant with segment I substituted had less specific activity against suc-AAPF- pNA compared to Savinase, whereas the Savinase variant with segment II substituted had more than 2 times higher specific activity against suc-AAPF-pNA than Savinase.
  • an AMSA-test (performed like described in Example 5 herein) the wash performance was shown to be preserved in Savinase variant with segment II compared to Savinase.
  • SEGMENT VIII S41 GTNFTD, positions 55-62 (SEQ ID NO: 40) Savinase VPG*EPST, positions 51-58 (SEQ ID NO: 41)
  • Savinase variants were constructed with the following segments from S41 : Segment X; Segments IX and X; Segments VIII and X; and Segments X and XI. All four Savinase variants showed subtilase activity on skim milk plates.
  • AMSA wash tests were performed on variants with Segment X and Segments X and XI like described in Example 5 herein.
  • the assay was conducted under the experimental conditions specified below:
  • the wash performance score (described in Example 5 herein) of the Savinase variants with Segment X and Segments X and XI was S (1) indicating an improved wash performance compared to Savinase.
  • HCIC Hydrophobic Charge Induction Chromatography
  • 4-MEP 4-Mercapto-Ethyl-Pyridine
  • Beads of the cellulose matrix sized 80-100 ⁇ m are mixed with a media containing yeast and the transformed B. subtilis capable of secreting the subtilisin variants and incubated at pH 9.5 in Unifilter ® microplates.
  • the concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST).
  • the purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites.
  • the protease and inhibitor binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive.
  • a substrate suc-AAPF-pNA 0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer
  • the degradation product pNA paranitrophenol
  • Wash performance of detergent compositions comprising modified enzymes Wash performance of detergent compositions comprising enzyme hybrids or enzyme variants of the present is tested at low washing temperature.
  • the Savinase variant Hybrid IV of Example 2 was tested for washing performance in two different assays; a microlitre scale assay (AMSA) and a millilitre scale assay (Mini wash).
  • AMSA microlitre scale assay
  • Mini wash millilitre scale assay
  • AMSA Automatic Mechanical Stress Assay
  • the performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific enzyme variant. Brightness can also be expressed as the intensity of the light reflected from the textile sample when luminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.
  • Colour measurements are made with a professional flatbed scanner (PFU DL2400pr ⁇ ), which is used to capture an image of the washed textile samples.
  • the scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits.
  • the scanner is frequently calibrated with a Kodak reflective IT8 target.
  • a special designed software application is used (Novozymes Color Vector Analyzer).
  • the program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB).
  • the intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
  • Int(v) is the light intensity value of textile surface washed with enzyme variant and lnt(r) is the light intensity value of textile surface washed with the reference enzyme subtilisin
  • Performance Scores are summing up the performances (P) of the tested enzyme variants as: S (2) which indicates that the variant performs better than the reference at all three concentrations (5, 10 and 30 nM) and
  • the millilitre scale wash performance assay was conducted under the following conditions:
  • the measurement of remission from the test material was done at 460 nm using a Zeiss MCS 521 VIS spectrophotometer. The measurements were done according to the manufacturer's protocol.
  • REMARK REFINEMENT REFINEMENT .
  • ATOM 236 N ASN A 16 13. .075 33 .432 21, .780 1. .00 17, .21 N
  • ATOM 238 CA ASN A 16 12. .534 34 .334 22, .811 1. .00 17 .53 C
  • ATOM 260 C ASP A 17 8, .937 35 .412 19, .778 1, .00 19 .80 C
  • ATOM 382 CA GLY A 26 24.296 29.380 10.938 1 1. . ,0000 1 155 . .4444 C
  • ATOM 422 C ILE A 29 28, .143 24 .586 9. .459 1, .00 14. .30 c
  • ATOM 462 N ALA A 32 26 .129 17 .789 11. .737 1, .00 14, .62 N
  • ATOM 471 O ALA A 32 25, .056 15, .556 13. .393 1, .00 12, .55 O
  • ATOM 507 N ASP A 35 21, .571 9, .765 12. .490 1. .00 11, .34 N
  • ATOM 518 O ASP A 35 20 .470 7. .623 11, .250 1, .00 12 .33 o
  • ATOM 558 CA TYR A 39 15. ,046 13. .247 3. 214 1. ,00 19. ,90 C
  • ATOM 604 CA HIS A 42 12 .245 17 .917 1, .419 1, .00 21, .28 C
  • ATOM 670 N ALA A 46 16 .104 21 .400 1 .079 1 .00 22 .51 N
  • ATOM 678 C ALA A 46 17. 880 22. .199 -0. 244 1. 00 20. ,28 c
  • ATOM 682 CA GLY A 47 20. ,217 22. .320 -0. ,968 1. ,00 21. .84 C
  • ATOM 703 N ALA A 49 22. ,035 19, .670 2. ,285 1. ,00 20. .96 N
  • ATOM 705 CA ALA A 49 22. ,951 18, .532 2. ,483 1. ,00 22. ,38 C
  • ATOM 730 CA GLN A 51 27, .273 15 .186 2, .847 1. .00 18, .38 C
  • ATOM 833 CD GLN A 57 23, .366 0, .800 2. .117 1. .00 36, .31 c
  • ATOM 842 CA SER A 58 26, .798 -4. .440 4. .371 1. .00 34, .96 C
  • ATOM 853 CA ASN A 59 29, .409 -2 .054 3, .003 1. .00 38, .41 C
  • ATOM 878 O PRO A 60 30 .266 2 .188 6 .749 1 .00 34 .22 O
  • ATOM 968 N ASP A 68 16. .070 8, .278 4. .623 1. .00 17 .54 N
  • ATOM 982 CA ARG A 69 12. .427 5, .549 5. .858 1. .00 20, .20 C
  • ATOM 984 CB ARG A 69 12. .665 4, .106 5. .357 1. .00 20, .57 C
  • ATOM 1020 O GLN A 70 9 .502 7. .745 10, .177 1, .00 18 .53 O
  • ATOM 1036 NDl HIS A 72 15, .356 10. .264 15. ,228 1. .00 11. .65 N
  • ATOM 1048 CA GLY A 73 16, .140 11, .851 11. .227 1. .00 13, .67 C
  • ATOM 1069 CA HIS A 75 12 .345 15 .653 11, ,803 1. .00 13, .53 C

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Abstract

The present invention relates to methods for producing variants of a parent TY145 subtilase and of a parent BPN' subtilase and to TY145 and BPN' variants having altered properties as compared to the parent TY145/BPN' subtilase.

Description

SUBTI ASES
FIELD OF THE INVENTION
The present invention relates to variants of TY145 subtilases and BPN' subtilases and to methods of construction such variants with altered properties, such as stability (e.g. thermo- stability or storage stability), Ca2+ dependency, pH dependent activity.
BACKGROUND OF THE INVENTION
Enzymes have been used within the detergent industry as part of washing formulations for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellulases, hemicellu- lases or mixtures of enzymes are also often used.
To improve the cost and/or the performance of proteases there is an ongoing search for proteases with altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc. The search for proteases with altered properties include both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g! genetic manipulation of the nucleic acid sequence encoding said proteases. Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific characteristic of the protein.
One family of proteases, which are often used in detergents, are the subtilases. This family has previously been further grouped into 6 different sub-groups by Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523. One of these sub-groups is the Subtilisin family which includes subtilases such as BPN', subtilisin 309 (SAVINASE®, NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE® NOVOZYMES A/S), subtilisin S41 (a subtilase from the psychrophilic Antarctic Bacillus TA41 , Davail S et al. 1994, The Journal of Biological Chemistry, 269(26), 99. 17448-17453), subtilisin S39 (a subtilase from the psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11), pp. 1271-1279) and TY145 (a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577). However, despite the sequence homology between the subtilases belonging to the Subtilisin subgroup of subtilases, modelling of the three-dimensional structure of one subtilase on the basis of the three-dimensional structure of another subtilase may result in an incorrect three- dimensional structure because of structural differences.
The inventors of the present invention have elucidated the three-dimensional structure of the TY 45 subtilase and found that there are several differences between this and the three- dimensional structure of BPN' also belonging to the Subtilisin subgroup of subtilases. This surprising difference in structure makes it advantageous to use the TY145 structure as basis for homology modelling of TY145 like subtilisins, which, in turn, will improve the ability to obtain desired changes in functionality by protein engineering.
Two studies have used protein engineering to alter functionality of TY145 like subtilisins: Mi- yazaki K et al. 2000, J Mol Biol, 297, pp.1015-1026 discloses enhancement of the thermostability and activity of the psychrophilic protease subtilisin S41 by methods of directed evolution.
Wintrode TL et al. 2000, Journal of Biological Chemistry, 275 (41), pp.31635-31640 discloses conversion of a mesophilic subtilisin-like protease from Bacillus sphaericus SSI I into its psy- chrophilic counterpart by methods of directed evolution. Wintrode et al. constructed the three-dimensional structural model of the SSII subtilase on basis of its homology with subtilisins Carlsberg, Savinase, BPN' and Thermitase. However, according to the present invention the SSII subtilase pertain to the new group of TY145 like subtilases and thus the modelling of SSII based on the 3D structure of the BPN' like subtilases will likely give an inaccurate result.
The differences between the three-dimensional structures of TY145 and BPN' are confirmed by the recently published three-dimensional structure of the subtilase "sphericase" from Bacillus sphaericus (PDB NO:1 EA7, Protein Data Bank). The overall structure and many details of this subtilase are very homologous to the TY145 subtilase structure.
BRIEF DESCRIPTION OF THE INVENTION
The inventors have modified the amino acid sequence of a subtilase to obtain variants with improved properties, based on the three-dimensional structure of the subtilases TY145 and BPN'. The variants have altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
Accordingly, the object of the present invention is to provide a method for constructing subtilases having altered properties, in particular to provide a method for constructing subtilases having altered properties as described above.
Thus, in its broadest aspect, the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: i) analyzing the three-dimensional structure of the subtilase to identify, on the basis of an evaluation of structural considerations, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; ii) constructing a variant of the subtilase, which as compared to the parent subtilase, has been modified in the amino acid residue or structural part identified in i) so as to alter said property; and iii) testing the resulting subtilase variant for said property.
Although it has been described in the following that modification of the parent subtilase in certain regions and/or positions is expected to confer a particular effect to the thus produced subtilase variant, it should be noted that modification of the parent subtilase in any of such regions may also give rise to any other of the above-mentioned effects. For example, any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability, may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased peptidase activity.
Further aspects of the present invention relates to variants of a subtilase, the DNA encoding such variants and methods of preparing the variants. Still further aspects of the present invention relates to the use of the variants for various industrial purposes, in particular as an additive in detergent compositions. Other aspects of the present invention will be apparent from the below description as well as from the appended claims.
BRIEF DESCRIPTION OF APPENDIX
APPENDIX 1 shows the structural coordinates for the solved crystal 3D structure of the TY145 subtilase.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows a multiple alignment of 3D sequences of subtilases from TY145, TA39,
TA41 , Bacillus sphaericus and Savinase.
Figure 2 shows an alignment between the amino acid sequences of subtilisin BPN' and Savinase in order to define the BPN' numbering of Savinase.
Figure 3 shows a superposition of TY145 subtilase (light) and BPN' structures (dark), with spheres indicating ion-binding sites. The TY145 ion-binding sites are light and the BPN' ion- binding sites are dark.
DEFINITIONS
Prior to discussing this invention in further detail, the following terms and conventions will first be defined. For a detailed description of the nomenclature of amino acids and nucleic acids, we refer to WO 00/71691 page 5, hereby incorporated by reference. A description of the nomenclature of modifications introduced in a polypeptide by genetic manipulation can be found in WO 00/71691 page 7-12, hereby incorporated by reference. The term "subtilases" refer to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue. Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
The Subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-S1 ("true" subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those de- scribed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
The term "parent" is in the context of the present invention to be understood as a protein, which is modified to create a protein variant. The parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means. For instance, the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide. Thus the term "parent subtilase" refers to a subtilase which is modified to create a subtilase variant.
The term "variant" is in the context of the present invention to be understood as a protein which has been modified as compared to a parent protein at one or more amino acid residues.
The term "modification(s)" or "modified" is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein. The modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest. Thus the term "modified protein", e.g. "modified subtilase", is to be understood as a protein which contains modification(s) compared to a parent protein, e.g. subtilase.
The term "(a) TY145 subtilase" or "(a) TY145 like subtilase" should in the context of the pre- sent invention be understood as a subtilase belonging to the Subtilisin group according to Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 63% homology to TY145, SEQ ID NO:1. In the context of the present invention a TY145 subtilase has three ion-binding sites.
The term "(a) BPN' subtilase" or "(a) BPN' like subtilase" should in the context of the present invention be understood as a subtilase belonging to the Subtilisin group according Siezen et al. Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 61% homology to BPN' SEQ ID NO:5. Such a BPN' like subtilase is for example Savinase. In the context of the present invention a BPN1 subtilase has two, three or five ion-binding sites. A BPN' like subtilase may, in the context of the present invention, belong to branch l-S of the subtilisins i.e. to branch I-S1, the "true" subtilisins or I-S2, the highly alkaline proteases (Siezen et al., Protein Engng. 4 (1991) 719-737).
"Homology" or "homologous to" is in the context of the present invention to be understood in its conventional meaning and the "homology" between two amino acid sequences should be determined by use of the "Similarity" defined by the GAP program from the University of Wisconsin Genetics Computer Group (UWGCG) package using default settings for alignment parameters, comparison matrix, gap and gap extension penalties. Default values for GAP penalties, i.e. GAP creation penalty of 3.0 and GAP extension penalty of 0.1 (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711). The method is also described in S.B. Nee- dleman and CD. Wunsch, Journal of Molecular Biology, 48, 443-445 (1970). Identities can be extracted from the same calculation. The homology between two amino acid sequences can also be determined by "identity" or "similarity" using the GAP routine of the UWGCG package version 9.1 with default setting for alignment parameters, comparison matrix, gap and gap extension penalties can also be applied using the following parameters: gap creation penalty = 8 and gap extension penalty = 8 and all other parameters kept at their default values. The output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" and the "Similarity" between the two sequences. The numbers calculated using UWGCG package version 9.1 is slightly different from the version 8.
The term "position" is in the context of the present invention to be understood as the number of an amino acid in a peptide or polypeptide when counting from the N-terminal end of said peptide/polypeptide. The position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to.
The four known subtilases belonging to the TY145 subgroup, i.e. subtilases obtained from TY145, TA39, TA41 and Bacillus sphaericus are numbered individually according to each of SEQ ID NO:1 ,2,3 and 4.
Likewise other subtilases belonging to the TY145 subgroup are numbered individually according to their own sequence. However in order to determine homologous positions in such other subtilases an alignment with each of SEQ ID's NO:1 ,2,3 and 4 is conducted according to the GAP procedure described above. Subsequently the homologous positions are determined with reference to the most homologous of SEQ ID's NO: 1 ,2,3 and 4. Alternatively subtilases belonging to the TY145 subgroup can be numbered by reference to the positions of TY145 subtilase (SEQ ID NO:1).
Subtilases belonging to the BPN' subgroup refers to the positions of Subtilisin Novo (BPN') from B. amyloliquefaciens (SEQ ID NO:5).
DETAILED DESCRIPTION OF THE INVENTION Despite the great homology of the subtilases described above the inventors of the present invention have elucidated the three-dimensional structure of TY145, SEQ ID NO:1 by X-ray crystallography and found that there are several substantial differences between the three dimensional structures of TY145 and BPN'. The inventors of the present invention have further compared the sequence homology of a representative number of subtilases belonging to the Subtilisin subgroup. This is shown in the homology matrix in Table 1 below.
Figure imgf000007_0001
Legend to Table 1 TY145 like subtilases:
1 : q45681 ; Subtilase derived from B. subtilis (BSTA41 ) 2: p28842; Psychrophilic subtilisin derived from Antarctic Bacillus strain (BSTA39)
3: abb77095; Subtilase derived from Bacillus sp. (TY145)
BPN' like subtilases, I-S1 :
4: p00783; Subtilase derived from Bacillus subtilis var. amylosacchariticus (BSAMY) 5: p29142; Subtilase derived from Bacillus stearothermophilus (BSSJ)
6: p35835; Subtilase derived from Bacillus subtilis var. natto. (BSNAT)
7: p07518; Subtilase derived from Bacillus pumilus (B. mesentericus) (BPMES)
8: p00782; Subtilase derived from Bacillus amyloliquefaciens (BPN')
9: p00780; Subtilase derived from Bacillus licheniformis (BLSCAR) BPN' like subtilases, I-S2
10: p41363; Subtilase derived from Bacillus halodurans (BHSAH)
11 : aaw62222; Subtilase derived from Bacillus lentus (BLS147)
12: p29600; Subtilase derived from Bacillus lentus (BLSAVI, BLS309)
13: p27693; Subtilase derived from Bacillus alcalophilus (BAALKP) 14: q99405; Subtilase derived from Bacillus sp. strain KSM-K16 (BSKSMK)
15: p29599; Subtilase derived from Bacillus lentus (BLSUBL).
On the basis of the 3D structure comparison and protein sequence the inventors of the present invention find that the subgroup of TY145 subtilases are different from BPN' subtilases based on the 3D structure comparison of the enclosed 3D structure of TY145 and the BPN' 3D structure but also indicated from the sequence homology between TY145 and BPN'.
TY145 subtilases
As described above a TY145 subtilase is in the context of the present invention to be under- stood as a subtilase which has at least 63% homology to SEQ ID NO:1. In particular said TY145 subtilase may have at least 65%, such as at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to TY145, i.e. to SEQ ID NO:1.
In a first embodiment of the present invention a TY145 subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of TY145, i.e. it may be homologous to the three-dimensional structure defined by the structure coordinates in Appendix 1. As it is well-known to a person skilled in the art that a set of structure coordinates for a protein or a portion thereof is a relative set of points that define a shape in three dimensions, it is possible that an entirely different set of coordinates could define an identical or a similar shape. Moreover, slight variations in the individual coordinates may have little or no effect on the overall shape.
These variations in coordinates may be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates of Appendix 1 (TY145 structure) may be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above. Alternatively, said variations may be due to differences in the primary amino acid sequence. If such variations are within an acceptable standard error as compared to the structure coor- dinates of Appendix 1 said three-dimensional structure is within the context of the present invention to be understood as being homologous to the structure of Appendix 1. The standard error may typically be measured as the root mean square deviation of e.g. conserved backbone residues, where the term "root mean square deviation" (RMS) means the square root of the arithmetic mean of the squares of the deviations from the mean. As it is also well-known to a person skilled in the art that within a group of proteins which have a homologous structure there may be variations in the three-dimensional structure in certain areas or domains of the structure, e.g. loops, which are not or at least only of a small importance to the functional domains of the . structure,, but which may result in a big root mean square deviation of the conserved residue backbone atoms between said structures.
Thus it is well known that a set of structure coordinates is unique to the crystallised protein. No other three dimensional structure will have the exact same set of coordinates, be it a homologous structure or even the same protein crystallised in different manner. There are natural fluctuations in the coordinates. The overall structure and the inter-atomic relationship can be found to be similar. The similarity can be discussed in terms of root mean square deviation of each atom of a structure from each "homologous" atom of another structure. However, only identical proteins have the exact same number of atoms. Therefore, proteins having a similarity below 100% will normally have a different number of atoms, and thus the root mean square deviation can not be calculated on all atoms, but only the ones that are considered "homologous". A precise description of the similarity based on the coordinates is thus difficult to describe and difficult to compute for homologous proteins. Regarding the present invention, similarities in 3D structure of different subtilases can be described by the content of homologous structural elements, and/or the similarity in amino acid or DNA sequence. For sequences having no deletions or insertions a RMS for the calcium atoms can be calculated.
Examples of TY145 like subtilases include the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41, herein also called TA41 subtilase (Davail S et al., 1994, J.
Biol. Chem., 269, 17448-17453), and the psychrophilic subtilisin protease S39 derived from the Antarctic Bacillus TA39, herein also called TA39 subtilase (Narinx E et al., 1997, Protein Engineering, 10 (11), 1271-1279). Recently a three-dimensional structure of a subtilisin homologous with the TY145 subtilisins was published in the Protein Data Bank (Accession No:1EA7). The overall structure and many details of this Bacillus sphaericus "sphericase" subtilase are very homologous with the TY145 subtilase structure; however the structure of the sphericase revealed as much as five ion-binding sites. The number of ion-binding sites may vary in similar structures depending on the medium used for crystallisation. Thus it appears that the two extra ion-binding sites of Bacillus sphaericus "sphericase" are due to a calcium containing crystallisation medium.
Accordingly, a preferred embodiment of the present invention is a parent subtilase or a subtilase variant which is at least 63% homologous to the sequence of SEQ ID NO:1 , preferably at least 65%, at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence of SEQ ID NO:1 , and optionally said subtilase further comprises the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites and wherein the Strong ion-binding site of the BPN' like subtilases is not present, and with the exception of the TY145 subtilase, the TA39 subtilase, the TA41 subtilase, and the Bacillus sphaericus "sphericase".
The TY145 subtilase of the present invention is encoded by an isolated nucleic acid sequence, which nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:20. Further the isolated nucleic acid sequence encoding a TY145 subtilase of the invention hy- bridizes with a complementary strand of the nucleic acid sequence shown in SEQ ID NO:20 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
Suitable experimental conditions for determining hybridization at *low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves pre- soaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-Iabeled (specific activity > 1 x 109 cpm/μg ) probe for 12 hours at ca. 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least * 55°C (low stringency), more preferably at least 60°C (medium stringency), still more preferably at least 65°C (medium/high stringency), even more preferably at least 70°C (high stringency), and even more preferably at least 75°C (very high stringency).
BPN' subtilases
As described above a BPN' subtilase is in the context of the present invention to be understood as a subtilase which has at least 61% homology to SEQ ID NO:5. In particular said BPN' subtilase may have at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to BPN', i.e. to SEQ ID NO:5.
In one embodiment of the present invention a BPN' subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of BPN'- as defined by the structure coordinates given, in PDB Nos. 1SBT and 1GNS (Protein Data Bank), or one of the several other structures of BPN' that are accessible from the Protein Data Bank. Variations between homologous structures may occur for several reasons as described above. Thus a BPN' subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the BPN' subti- lases as described above, and in addition such subtilases does preferably not have further structural characteristics which are not present in the BPN1 subtilases as described herein. Further a BPN' subtilase of the present invention may have the necessary percentage of similarity with SEQ ID NO:5.
Examples of BPN' like subtilases include the subtilisin 309 (PDB NO:1SVN SAVINASE®, NOVOZYMES A/S) and subtilisin Carlsberg (ALCALASE®, NOVOZYMES A/S), among others.
In figure 1 of R.J. Siezen and J.A.M Leunissen (Protein science, Vol. 6 (3), pp. 501-523, 1997) page 502 a structure of subtilases is described. A subtilase consists of 6-8 helices, 11 strands of which 7 are central in a twisted beta-sheet. Two ion-binding sites are mentioned, one of which is the so called "Weak" calcium-binding site. It was later discovered that for some structures (subtilisin DY PDB no. 1 BH6, 1998), this calcium-binding site was shown to be a Na (sodium) binding site when the calcium concentration in the crystallization medium was low. Thus, in the following we refer to ion-binding sites instead of calcium-binding sites.
The BPN' subtilase of the present invention is encoded by an isolated nucleic acid sequence, which nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:21. Further the isolated nucleic acid sequence encoding a BPN' subtilase of the invention hybridizes with a complementary strand of the nucleic acid sequence shown in SEQ ID NO:21 preferably under low stringency conditions, but at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
Three-dimensional structure of TY145 subtilases The TY145 subtilase was used to elucidate the three-dimensional structure forming the basis for the present invention.
The structure of TY145 was solved in accordance with the principle for x-ray crystallographic methods, for example, as given in X-Ray Structure Determination, Stout, G.K. and Jensen, L.H., John Wiley & Sons, Inc. NY, 1989. The structural coordinates for the solved crystal structure of TY145 are given in standard PDB format (Protein Data Bank, Brookhaven National Laboratory, Brookhaven, CT) as set forth in Appendix 1. It is to be understood that Appendix 1 forms part of the present application. In the context of Appendix 1 , the following abbreviations are used: CA refers to c-alpha (carbon atoms) or to calcium ions, (however to avoid misunderstandings we use the full names "c-alpha atoms" and "calcium" or "ion" in the present specification). Amino acid residues are given in their standard three-letter code. The attached structural coordinates contain the protease structure, and an inhibitor structure CI2 as well as water molecules. The protease coordinates has a chain identification called A, whereas the CI2 inhibitor is called B, the calcium ions are called C, and the water is W. In the following the positions of the men- tioned residues refer to the sequence of TY145 as disclosed in SEQ ID NO:1.
The structure of TY145 shows the same "overall" fold as found in the S8 family of subtilisins. The structure comprises a twisted beta-sheet with 7 strands arranged in the following sequential order S2, S3, S1 , S4, S5, S6, S7. There are six alpha helices in the structure of which number H1 contains residues 9-15, H2 contains residues 72-81 , H3 contains residues 114-131 , H4 contains residues 148-158, H5 contains residues 250-267and H6 contains residues 273-286. The TY145 like subtilases are shown to lack the well-known Strong ion-binding site of the BPN' subtilases. However, in addition to the Weak calcium or ion-binding site also known from the BPN' subtilases, the TY145 subtilases have two ion-binding sites which are not present in the BPN' subtilisin structures. This can be seen in the structural alignment presented in Figure 3. These additional ion-binding sites are hereinafter referred to as "Near" and "Far" according to their distance to the Weak ion-binding site. Thus in relation to the atomic coordinates disclosed in Appendix 1 , the ion-binding sites of TY145 are located at: Weak - calcium atom named C 314, Near - calcium atom named C 312, and Far - calcium atom named C 313 in the PDB table (Appendix 1).
The position of an ion-binding site can be defined by the distance to four specific atoms in the core structure. The distance from the ion-binding site to the c-alpha atoms of the three active site residues has been chosen. Throughout the subtilases the residues Ser, His and Asp in the active site are highly conserved. In TY145 they are Asp35, His72 and Ser251. The fourth distance chosen is the distance to the c-alpha atom of the amino acid residue coming first after the active site serine residue in the sequence (herein after called "next to Ser"); in the 3D structure of TY145 it is Met252. • .
In a preferred embodiment of the present invention, the distance between: a) the Weak ion-binding site and i) Asp c-alpha atom is 17.50-19.50A, ii) His c-alpha atom is 21-23A, iii) Ser c-alpha atom is 13.80-15.80A, iv) next to Ser c-alpha atom is 15.80-
17.80A, b) the Far ion-binding site and i) Asp c-alpha atom is 28.70-30.70A, ii) His c-alpha atom is
28-30A, iii) Ser c-alpha atom is 20-22A, iv) next to Ser c-alpha atom is 19.50-21.50A, c) the Near ion-binding site and i) Asp c-alpha atom is 27-29A, ii) His c-alpha atom is
29.50-31.50A, iii) Ser e-alpha atom is 21.40-23.40A, iv) next to Ser e-alpha atom is
22.50-24.50A.
Below are the specific distances between the four chosen c-alpha atoms and the three ion binding sites of the TY145 subtilase given in A:
Weak ion-binding site Far ion-binding site Near ion-binding site
Met252 c-alpha atom 16.75 20.35 23.58
His72 c-alpha atom 21.98 29.10 30.43
Asp35 c-alpha atom 18.55 29.68 28.04 Ser251 c-alpha atom 14.71 20.96 22.28
Weak ion-binding site 0 16.62 9.79
Far ion-binding site 16.62 0 12.48
Near ion-binding site 9.79 12.48 0 However these distances may vary from one subtilase to the other, and as described above, the Weak ion binding site may also bind to a sodium ion. The present distances are given with a calcium ion in the structure. If a sodium ion was bound instead the distances would be shifted a little bit. Generally the distances can vary ±O.δA, preferably +0.7A, ±0.6A, ±O.δA, ±0.4A, or most preferably ±0.3A.
Further, in the TY145 like subtilases, the peptide structure circumscribing the Weak ion- binding site is composed of the amino acid residues placed in positions 182-189 and 221- 227 with the coordinating atoms being the backbone carbonyl oxygen atom of residues G182, A187, L184 and two water molecules.
The peptide structure circumscribing the Near ion-binding site is composed of residues 212- 225 with the coordinating atoms being the backbone carbonyl oxygen atom of residues I220 and T215, the oxygens from the carboxylic acids of residues D225 and D218 and the amid group of residue Q222. The peptide structure circumscribing the Far ion-binding site is composed of residues 288- 306 with the coordinating atoms being the backbone carbonyl oxygen atom of residues G298, G296 and I289, the oxygens from the carboxylic acids of residues D300 and D288, and two water molecules.
In comparison with the BPN' like subtilase structures the structure of the TY145 like subtilase can be divided into a "common subtilase-like" region, an "intermediate" region and a "non- homolo-gous" region.
The active site can be found in the common subtilase-like region, which is structurally closely related to the BPN' structures. The common subtilase-like region is composed of residues 88-128 and 225-284, and contains the alpha-helix H3 and the central alpha-helix H5 in which the active site serine residue is situated in the N-terminal part. The common subtilase-like region has an RMS lower than 1.2.
Outside the common subtilase-like region the structure of the TY145 like subtilase differs from the BPN' structures to a greater extent.
The intermediate region consist of residues 24-45, 48-58, 65-66, 67-85, 134-174, 175-196, 202-212 and 287-290. The intermediate region has an RMS higher than 1.2 and lower than 1.8. The relationships between the three-dimensional structure and functionality are potentially difficult to predict in this region of the TY145 like subtilases.
The nonhomologous region consists of residues 5-15, 16-23, 86-87, 129-133, 197-201 , 213-
124, 285-286, 291-298 and 299-311. The nonhomologous region has a RMS higher than 1.5, which also pertains to residues 65-66 from the intermediate region. The group comprising residues 5-15 and 299-311 has an RMS between 2.1-2.2. The relationships between the three-dimensional structure and functionality are very difficult to predict in this region of the TY145 like subtilases.
The regions in areas A1-T5, N16-T24, A46-Q51 , S58-C66, G84-G90, S129-K134, S129- K134, S173-S175, V196-T201 , N212-R224, A284-V286, K290-D299 and V310-K311 in the TY145 structure differs significantly from the other S8 family subtilisins (including the BPN1 type subtilisins) in c-alpha atom coordinates. An RMS cannot be calculated for these last residues as there are no homologous c-alpha atoms in the compared subtilases.
Homology building of TY145 and BPN' like subtilases
A model structure of a TY145 like subtilase or a BPN' like subtilase can be built using the Homology program or a comparable program, e.g., Modeller (both from Molecular Simulations, Inc., San Diego, CA). The principle is to align the amino acid sequence of a protein for which the 3D structure is known with the amino acid sequence of a protein for which a model 3D structure has to be constructed. The structurally conserved regions can then be built on the basis of consensus sequences. In areas lacking homology, loop structures can be inserted, or sequences can be deleted with subsequent bonding of the necessary residues using, e.g., the program Homology. Subsequent relaxing and optimization of the structure should be done using either Homology. or another molecular simulation, program," e.g., CHARMm from Molecular Simulations.
Methods for designing TY145 and Subtilisin family subtilase variants
Comparisons of the molecular dynamics of different proteins can give a hint as to which do- mains are important or connected to certain properties pertained by each protein.
The present invention comprises a method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 subtilase on the three-dimensional structure of a TY145 subtilase to produce a three-dimensional structure of the parent TY145 subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent TY145 subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions corresponding to said structural part; e) expressing the modified nucleic acid sequence in a host cell to produce the variant
TY145 subtilase; f) isolating the produced subtilase; g) purifying the isolated subtilase and h) recovering the purified subtilase.
Further the present invention comprises a method of producing a variant of a parent Subtil- isin family subtilase, such as a BPN' like subtilase, the variant having at least one altered property as compared to the parent Subtilisin family subtilase, the method comprising: a) modelling the parent Subtilisin family subtilase on the three-dimensional structure of a Subtilisin family subtilase to produce a three-dimensional structure of the parent Subtilisin family subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 like subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of , the parent Subtilisin family subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent Subtilisin family subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions corresponding to said structural part; e) expressing the modified nucleic acid sequence in a host cell to produce the variant Sub- tilisin family subtilase, f) isolating the produced subtilase, g) purifying the isolated subtilase and h) recovering the purified subtilase.
Further the present invention comprises a method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 like subtilase on the three-dimensional structure of a TY145 like subtilase to produce a three-dimensional structure of the parent TY145 like subti- lase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a Subtilisin family subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 like subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent TY145 like subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino ac- ids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions corresponding to said structural part; e) expressing the modified nucleic acid sequence in a host cell to produce the variant TY145 like subtilase; f) isolating the produced subtilase; g) purifying the isolated subtilase and h) recovering the purified subtilase.
Stability - alteration of ion-binding sites
As described above the TY145 subtilases has two new ion-binding sites not present in the BPN' subtilisin structures but lacks the Strong ion-binding site of the BPN' subtilases. Stability of the ion-binding site is of crucial importance for the functionality of the enzyme. Therefore alterations of the amino acid residues close to the ion-binding sites are likely to result in alterations of the stability of the enzyme. The positions which may be modified are located: Weak: at a distance of 10A or less around calcium atom named C 314, Near: at a distance of 10A or less around calcium atom named C 312, and Far: at a distance of 10A or less around calcium atom named C 313 in the PDB table (Appendix 1).
Improved stability
Stabilisation of the ion-binding sites of TY145 may possibly be obtained by alterations in the positions close to the sites. Positions located at a distance of 10A or less to the ion-binding sites of TY145 (SEQ ID NO:1) are:
Weak: 154, 155, 158, 164, 165, 166, 167, 168, 178-191 (i.e. 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191), 211 , 220-228 (i.e. 220, 221 , 222, 223, 224, 225,
226, 227, 228), 277, 281 and 305.
Near: 185, 211-227 (i.e. 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227), 277, 281, 299, 300, 301 , 304, 305.
Far: 193, 198, 199, 201 , 202, 204, 216, 217, 219, 226, 227, 228, 229 and 284-307 (i.e. 284,
285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302,
303, 304, 305, 306, 307). In detergent compositions calcium chelaters contribute to removal of calcium from the subtilases with subsequent inactivation of the enzyme as the result. To decrease the inactivation due to calcium removal of e.g. calcium chelaters, variants with improved calcium stability can be constructed. Variants with alterations close to the Near ion-binding site are I220S,T and T215S, variants with alterations close to the Far ion-binding site are G298A,S,T and G296A,S,T, and variants with alterations close to the Weak ion-binding site are V185T and I221N,D,T.
TY145 with extra ion-binding site The Strong ion-binding site from the BPN' subtilases can be transplanted into TY145 (or other subtilases in TY145 subgroup) by deletion(s) of or in the region H83-G90 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues. A preferred variant has the whole region deleted and a subsequent insertion between A82 and V91 of the sequence LNNSIG.
Removal of ion-binding site in TY145
By removing a ion-binding site it is possible to alter the enzymes dependency of calcium or other ions in the solution. The Far and Near ion-binding sites in TY145 (or others from TY145 group) can be removed with guidance from the three-dimensional structure of BPN' and Sav-. inase (or others in BPN1 group).
Removal of the Far site can be done by deletion(s) of or in the region K290-D300 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues. A preferred variant has the whole region deleted and a subsequent insertion between I289 and Y301 of the sequence GDS or DST. Preferably, but not mandatory the substitution S303Y is further added. Removal of the Near site can be done by deletion(s) of or in the region N212-R224 (of SEQ ID NO:1) and subsequent insertion of one or more amino acid residues. A preferred variant has the whole region deleted and a subsequent insertion of a proline or alanine residue between G211 and D225.
Removal of Strong ion-binding site in BPN' subtilases
The Strong ion-binding site in BPN' like subtilases can be removed. Exemplified in Savinase, the removal can be done by deletion of or in the region L75-G80 (BPN' numbering) and subsequent insertion of one or more amino acid residues. A preferred variant has the whole re- gion deleted and a subsequent insertion of residues 84-88 from TY145. In addition the substitutions L82Y and Q2A,N can be applied. Alteration of thermostability
A variant with improved stability (typically increased thermostability) may be obtained by substitution with proline, introduction of a disulfide bond, altering a hydrogen bond contact, altering charge distribution, introduction of a salt bridge, filling in an internal structural cavity with one or more amino acids with bulkier side groups (in e.g. regions which are structurally mobile), substitution of histidine residues with other amino acids, removal of a deamidation sites, or by helix capping.
Regions with increased mobility: The following regions of TY145 have an increased mobility in the crystal structure of the enzyme, and it is presently believed that these regions can be responsible for stability or activity of TY145. Especially thermostabilisation may possibly be obtained by altering the highly mobile regions. Improvements of the enzyme can be obtained by mutation in the below regions and positions. Introducing e.g. larger residues or residues having more atoms in the side chain could increase the stability, or, e.g., introduction of residues having fewer atoms in the side chain could be important for the mobility and thus the activity profile of the enzyme, The regions can be found by analysing the B-factors taken from the coordinate file in Appendix 1 , and/or from molecular dynamics calculations of the isotropic fluctuations. These can be obtained by using the program CHARMm from MSI (Molecular Simulations Inc.).
Molecular dynamics simulation at 300K of TY145 reveals the following highly mobile regions:
84-89 (i.e. 84, 85, 86, 87, 88)
108-117 (i.e. 108, 109, 110, 111, 112, 113, 114, 115, 116, 117)
141-146 (i.e. 141 , 142, 143, 144, 145, 146) 150-152 (i.e.150, 151 , 152) 169-171 (i.e. 169, 170, 171) 200-201
211-220 (i.e. 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220) 242-243 268-270 (i.e. 268, 269, 270).
Also B-factors (see "in X-Ray Structure Determination, Stout, G.K. and Jensen, L.H., John
Wiley & Sons, Inc. NY, 1989") from crystallographic data indicates the following more mobile regions in the TY145 structure: 1-7 (i.e. 1 , 2, 3, 4, 5, 6, 7),
17-23 (i.e. 17, 18, 19, 20, 21 , 22, 23),
38-50 (i.e. 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50),
57-69 (i.e. 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69), 84-92 (i.e. 84, 85, 86, 87, 88, 89, 90, 91, 92),
107-110 (i.e. 107, 108, 109, 110), 239-243 (i.e. 239, 240, 241 , 242, 243) and 265-266. Preferably the regions 57-69 and 84-92.
Disulfide bonds:
A TY145 variant of the present invention with improved stability, e.g. thermostability, as compared to the parent TY145 may be obtained by introducing new inter-domain or intra-domain bonds, such as by establishing inter- or intra-domain disulfide bridges.
Thus a further aspect of the present invention relates to a method for producing a variant of a parent TY145 comprising the methods described in the paragraph "Methods of preparing TY145 like or BPN' like subtilase variants" herein.
According to the guidelines mentioned above the below mentioned amino acid residues iden÷ tified in the amino acid sequence of SEQ ID NO:1 are contemplated as being suitable for cysteine replacement. With one or more of these substitutions with cysteine, disulfide bridges may possibly form in a variant of TY145. The substitutions are: G26C+A95C; A167C+T254C; R203C+G292C and V228C+A284C:
Similar residues suitable for cysteine replacement in homologous subtilases such as TA39, TA41 can be elucidated by finding the homologous positions in the alignment of Figure 1. Concerning another TY145 like sequence the homologous positions suitable for cysteine replacement can be selected by aligning said TY145 like sequence with all of the sequences of Figure 1 using the GAP analysis method as described above. The suitable residues can then be selected in accordance with the homologous positions in the most homologous of SEQ ID's NO:1 ,2,3 and 4 which are the sequences of the subtilases aligned in Figure 1.
Surface charge distribution A variant with improved stability (typically improved thermostability) as compared to the parent subtilase may be obtained by changing the surface charge distribution of the subtilase. For example, when the pH is lowered to about 5 or below histidine residues typically become positively charged and, consequently, unfavorable electrostatic interactions on the protein surface may occur. By engineering the surface charge of the subtilase one may avoid such unfavorable electrostatic interactions that in turn lead to a higher stability of the subtilase.
Therefore, a further aspect of the present invention relates to a method for constructing a variant of a parent subtilase, the method comprising: a) identifying, on the surface of the parent subtilase, preferably a TY145 like or a BPN' like subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one amino acid residue se- lected from the group consisting of Asp, Glu, Arg, Lys and His with an uncharged amino acid residue; c) optionally repeating steps a) and b) recursively; d) optionally, making alterations each of which is an insertion, a deletion or a substitution of an amino acid residue at one or more positions other than b); e) preparing the variant resulting from steps a) - d); f) testing the stability of said variant; and g) optionally repeating steps a) - f) recursively; and h) selecting a subtilase variant having increased stability as compared to the parent subtilase.
As it will be understood by the skilled person it may also, in some cases, be advantageous to substitute an uncharged amino acid residue with an amino acid residue bearing a charge or, alternatively, it may in some cases be advantageous to substitute an amino acid residue bearing a charge with an amino acid residue bearing a charge of opposite sign. Thus, the above-mentioned method may easily be employed by the skilled person also for these purposes. In the case of substituting an uncharged amino acid residue with an amino acid residue bearing a charge the above-mentioned method may be employed the only difference being steps a) and b) which will then read: a) identifying, on the surface of the parent subtilase, at least one uncharged amino acid residue; b) substituting, on the surface of the parent subtilase, at least one uncharged amino acid residue with a charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His.
Also in the case of changing the sign of an amino acid residue present on the surface of the subtilase the above method may be employed. Again, compared to the above method, the only difference being steps a) and b) which, in this case, read: a) identifying, on the surface of the parent subtilase, at least one charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one charged amino acid resi- due identified in step a) with an amino acid residue having an opposite charge.
Thus, Asp may be substituted with Arg, Lys or His; Glu may be substituted with Arg, Lys or
His; Arg may be substituted with Asp or Glu; Lys may be substituted with Asp or Glu; and His may be substituted with Asp or Glu.
In order to determine the amino acid residues of a subtilase, which are present on the surface of the enzyme, the surface accessible area are measured using the DSSP program (Kabsch and Sander, Biopolymers (1983), 22, 2577-2637). All residues having a surface ac- cessibilty higher than 0 is regarded a surface residue.
An amino acid residue found on the surface of TY145 using the above method is D116 and it is contemplated that the substitutions D116H,K,R are of particular interest.
Similar substitutions may be introduced in equivalent positions of other TY145 like subtilases.
Substitution with proline residues
Improved thermostability of a subtilase can be obtained by subjecting the subtilase in question to analysis for secondary structure, identifying residues in the subtilase having dihedral angles φ (phi) and ψ (psi) confined to the intervals [-90°<φ<-40° and -180°<ψ<180°], preferably the intervals [-90°<φ<-40° and 120°<ψ<180°] or [-90°<φ<-40° and -50°<ψ<10°] and excluding residues located in regions in which the subtilase is characterized by possessing α-helical or β-sheet structure.
After the dihedral angles φ (phi) and ψ (psi) for the amino acids have been calculated, based on the atomic structure in the crystalline subtilases, it is possible to select position(s) which has/have dihedral phi and psi angles favourable for substitution with a proline residue. The aliphatic side chain of proline residues is bonded covalently to the nitrogen atom of the peptide group. The resulting cyclic five-membered ring consequently imposes a rigid constraint on the rotation about the N-Cα bond of the peptide backbone and simultaneously prevents the formation of hydrogen bonding to the backbone N-atom. For these structural reasons, proline residues are generally not compatible with α-helical and β-sheet secondary conformations. If a proline residue is not already at the identified position(s), the naturally occurring amino acid residue is substituted with a proline residue, preferably by site directed mutagenesis applied on a gene encoding the subtilase in question.
In the group of TY145 like subtilases proline residues can be introduced at positions 18, 115, 185, 269 and 293. Accordingly, a preferred TY145 variant has one or more of the substitu- tions: Q18P, D115P, V185P, T269P and I293P.
Alteration of activity
Introduction of activity at low temperature in TY145 and Savinase
A comparison of the molecular dynamics at 300K of TY145 (a mesophilic-derived enzyme obtained from crystal structure) and TA41 (a psychrophilic derived enzyme obtained from modelling) was conducted. The comparison was directed to low temperature activity and revealed a difference in dy- namical behaviour of TY145 and TA41. The theory derived from the comparison is that the difference in dynamics, especially around the active site, are important for the low temperature functionality of the psychrophilic enzyme. The necessary dynamics are needed for the enzyme to have activity at low temperature and thus the activity drops if the enzymes dynam- ics are lowered.
The higher mobility regions in TA41 compared to TY145 measured by molecular dynamics simulation indicates important regions for the low temperature activity, of the enzyme TA41 which can be transferred to TY145. The regions in TA41 are: 16-22 (i.e. 16, 17, 18, 19, 20, 21, 22),
40-73 (i.e. 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73), 118-131 (i.e.118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131) 140-161 (i.e.140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161) and
275-294 (i.e.275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291,292,293,294).
Regions closest to the active site and the substrate binding site are regarded as preferred in relation to making higher activity at low temperature for TY145: 40-73, and 140-161 , preferably 65-73 and 140-150. The regions in TY145 should be modified to be more mobile for example by substitution with small less rigid residues, i.e. residues with smaller side chains (such as Gly, Ala, Ser, Thr or Val), into the TY145 backbone.
The other regions in TA41 are most interesting for stabilisation of the psychrophilic enzyme. These regions can easily be found in TA39 as well or in other homologous enzymes, also non psychrophilics.
The regions around the active site and the substrate binding site are the regions most likely involved in the low temperature functionality. Below are suggestions for transferring the low temperature activity of TA41 and homologous sequences to TY145-like sequences and the BPN'-like sequences:
TA41 TY145 Savinase
131 V31 I,A,L V28l,A,L
V38 V38A.L I35V,A,L
S79 T79S T71S
A80 V80A,G,V I72A,G,V
L81 L81G A73L,G V187 V188A M175V,A
T253 T254S.A T224S.A
The numbering is according to SEQ ID NO:3, 1 and 5 respectively. Savinase is numbered according to BPN'. Preferred Savinase variants are V28l, I35V, T71S, I72A, A73L, M175V and T224S. Examples of core variants of TY145 are: V31 I, V80A, T79S.
The alterations of the TY145-like sequences and the BPN'-like sequences can be single mutations or combinations of the suggested mutations.
Substrate bindings site
The substrate binding site is identified by the residues in contact with a substrate model, such as the CI2 inhibitor. The 3D structure coordinates of the TY145 subtilase with CI2 bound in the active site can be found in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favor- able interactions found within a sphere 10 A from the substrate molecule, in particular within a sphere of 6 A from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions. The following residues of the TY145 subtilase (SEQ ID NO:1), are within a distance of 6A from the CI2 inhibitor and thus believed to be involved in interactions with said substrate: 35, 36, 70, 72, 106, 109, 110, 111 , 112, .113, 114, 117, 139, 140, 141 , 142, 143, 144, 145, 147, 150, 167, 168, 169, 170, 171 , 172, 173, 174, 177, 180, 207, 239, 247, 148, 149, 150, 151 and 252.
Stabilization by modification of Asn-Gly pairs It is known that at alkaline pH, the side chain of Asn may interact with the NH group of a sequential neighbouring amino acid to form an isoAsp residue where the backbone goes through the Asp side chain. This will leave the backbone more vulnerable to proteolysis. The deamidation is much more likely to occur if the residue that follows is a Gly. Changing the Asn in front of the Gly or the Gly will prevent this from happening and thus improve the stabil- ity, especially as concerns thermo- and storage stability.
The invention consequently further relates to a subtilase, in which either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent RP-II protease is/are deleted or substituted with a residue of a different amino acid. The Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
Asn-Gly sequences can be found in the following positions: B. sphaericus: 198-199, 240-241 TY145: 87-88, 109-110, 199-200 TA41 : 83-84, 198-199 TA39: 88-89, 198-199
The present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
Modification of Tyrosine residues In relation to wash performance it has been found that the modification of certain tyrosine residues to phenylalanine provides an improved wash performance. Without being bound by any specific theory, it is believed that titration of these Tyr residues in the alkaline wash liquor has negative effects that are alleviated by replacing the Tyr residues with other residues, especially Phe or Trp, particularly Phe.
Tyrosines can be found in the following positions:
B. sphaericus: 14, 91 , 102, 112, 155, 157, 172, 179, 201 , 206, 211, 218, 235, 239, 243, 292,
300,
TY145: 15, 39, 92, 103, 113, 156, 158, 202; 219, 240, 244, 287, 301 , 307, TA41 : 15, 91, 102, 112, 155, 157, 179, 201 , 218, 235, 243,
TA39: 15, 61 , 91 , 102, 112, 155, 157, 173, 179, 201 , 211, 218, 235, 243, 267, 281 , 284, 292, 293, 296
The present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
Modification of methionine residues
In order to improve the oxidation stability of proteins it has been found that the substitution or even deletion of methionine residues is beneficial, Especially modification of the methionine residue normally found next to the active serine residue may provide a significant improvement of the oxidation stability. Modifications to Ser or Ala are the most preferred substitutions for this Met.
Methionines can be found in the following positions: B. sphaericus: 138, 251 ,
TY145: 139, 252,
TA41 : 1 , 138,251 ,
TA39: 1 , 138, 251 , The present invention in this respect thus relates to modifications, such as deletions and substitutions in one or more of these positions in accordance with the principles given above.
Combined modifications
The present invention also encompasses any of the above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged. Such combinations comprise the positions: 222 (improves oxidation stability), 218 (improves thermal stability), substitutions in the Ca2+-binding sites stabilizing the enzyme, e.g. position 76, and many other apparent from the prior art (all positions according to BPN' numbering).
In further embodiments a subtilase variant described herein may advantageously be com- bined with one or more modification(s) in any of the positions:
27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 120, 123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (BPN' numbering).
Specifically, the following BLSAVl, BLSUBL, BSKSMK, and BAALKP modifications are considered appropriate for combination:
K27R, *36D, S56P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N, V104Y, H120D, N123S, G159D, Y167A, R170S, R170L, Q206E, N218S, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A (BPN* numbering).
Furthermore variants comprising any of the modifications S101G+V104N,
S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I or
N76D+V104A, or other combinations of the modifications K27R, N76D, S101G, S103A, V104N, V104Y, V104I, V104A, N123S, G159D, A232V, Q236H, Q245R, N248D, N252K,
T274A in combination with any one or more of the modification(s) mentioned above exhibit improved properties.
A particular interesting variant is a variant, which, in addition to modifications according to the invention, contains the following substitutions: S101 G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.
Moreover, subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, 131 and 194, preferably as 129K, 131 H and 194P modifications, and most preferably as P129K, P131 H and A194P modifications. Any of those modification(s) are expected to provide a higher expression level of the subtilase variant in the production thereof.
Methods of preparing TY145 like or BPN' like subtilase variants
The subtilase variants, i.e. the TY145 and BPN' variants of the present invention may be produced by any known method within the art and the present invention also relates to nucleic acid encoding a subtilase variant of the present invention, a DNA construct comprising said nucleic acid and a host cell comprising said nucleic acid sequence. In general natural occurring proteins may be produced by culturing the organism expressing the protein and subsequently purifying the protein or it may be produced by cloning a nucleic acid, e.g. genomic DNA or cDNA, encoding the protein into an expression vector, introducing said expression vector into a host cell, culturing the host cell and purifying the expressed protein. Typically protein variants may be produced by site-directed mutagenesis of a parent protein, introduction into expression vector, host cell etc. The parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof. In general standard procedures for cloning of genes and/or introducing mutations (random and/or site directed) into said genes may be used in order to obtain a parent subtilase, or subtilase or subtilase variant of the invention. For further description of suitable techniques reference is made to Molecular cloning: A laboratory manual (Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.)); Current protocols in Molecular Biology (John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)); Molecular Biological Methods for Bacillus (John Wiley and Sons, 1990); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); A Practical Guide To Molecular Cloning (B. Perbal, (1984)) and WO 96/34946.
Further, variants could be constructed by:
Random Mutagenesis Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.
The random mutagenesis of a DNA sequence encoding a parent subtilase may be conven- iently performed by use of any method known in the art.
In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a parent subtilase, wherein the variant exhibits an altered property, such as increased thermostability, increased stability at low pH and at low calcium concentration, relative to the parent subtilase, the method comprising:
(a) subjecting a DNA sequence encoding the parent subtilase to random mutagenesis,
(b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and
(c) screening for host cells expressing a subtilase variant which has an altered property relative to the parent subtilase. Step (a) of the above method of the invention is preferably performed using doped primers. For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents. The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS); sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties. When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions that are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the subtilase enzyme by any published tech- nique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate. Preferably, the doping is carried out using "constant random doping", in which the percentage of wild-type and modification in each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues. The doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% modifications in each position. An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided (L.J. Jensen et al. Nucleic Acid Research, 26, 697-702 (1998).
When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent subtilase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, 1 , 1989, pp. 11-15). A mutator strain of £. coli (Fowler et al., Molec. Gen. Genet, 133, 1974, 179-191), S. cere- viseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the subtilase by, e.g., transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may be subsequently transformed into the ex- pression organism.
The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent subtilase. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenising agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harbored in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence. In some cases it may be convenient to amplify the mutated DNA sequence prior to perform- ing the expression step b) or the screening step c). Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR- generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme. Subsequent to the incubation with or exposure to the mutagenising agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermo- philus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circu- lans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gram negative bacteria such as E. coli. The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.
Localised random mutagenesis
The random mutagenesis may be advantageously localised to a part of the parent subtilase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
The localised or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
General method for random mutagenesis by use of the DOPE program The random mutagenesis may be carried out by the following steps: 1. Select regions of interest for modification in the parent enzyme
2. Decide on mutation sites and non-mutated sites in the selected region
3. Decide on which kind of mutations should be carried out, e.g. with respect to the de sired stability and/or performance of the variant to be constructed
4. Select structurally reasonable mutations 5. Adjust the residues selected by step 3 with regard to step 4.
6. Analyse by use of a suitable dope algorithm the nucleotide distribution.
7. If necessary, adjust the wanted residues to genetic code realism, e.g. taking into account constraints resulting from the genetic code, e.g. in order to avoid introduction of stop codons; the skilled person will be aware that some codon combinations can- not be used in practice and will need to be adapted
8. Make primers
9. Perform random mutagenesis by use of the primers
10. Select resulting subtilase variants by screening for the desired improved properties.
Suitable dope algorithms for use in step 6 are well known in the art. One such algorithm is described by Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11 :29- 38. Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A, and Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).
Expression vectors
A recombinant expression vector comprising a nucleic acid sequence encoding a subtilase variant of the invention may be any vector that may conveniently be subjected to recombinant DNA procedures and which may bring about the expression of the nucleic acid se- quence.
The choice of vector will often depend on the host cell into which it is to be introduced. Examples of a suitable vector include a linear or closed circular plasmid or a virus. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβl Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehr- lich, 1978, Proceedings of the National Academy of Sciences USA 75:1433). Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome, in particular it may contain nucleic acid, sequences facilitating integration into the genome by homologous or non-homologous recombination. The vector system may be a single vector, e.g. plasmid or virus, or two. or more vectors; e.g. plasmids or virus', which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
The vector may in particular be an expression vector in which the DNA sequence encoding the subtilase variant of the invention is operably linked to additional segments or control sequences required for transcription of the DNA. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence encoding the subtilase variant. Additional segments or control sequences include a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum the control sequences include a promoter and transcriptional and translational stop signals.
The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for use in bacterial host cells include the promoter of the Ba- cillus subtilis levansucrase gene (sacB), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amylolique- faciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacil- lus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731). Other examples include the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters or the Streptomyces coelicolor agarase gene (dagA). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra. Examples of suitable promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable al- pha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusahum oxysporum trypsin-like protease (as described in U.S. Patent No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters. Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpiA promoter. Examples of suitable promoters for use in yeast host cells include promoters from yeast gly- colytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters. Further useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423- 488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
Examples of suitable promoters for use in mammalian cells are the SV40 promoter (Subra- mani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
An example of a suitable promoter for use in insect cells is the polyhedrin promoter (US
4,745,051 ; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
The DNA sequence encoding a subtilase variant of the invention may also, if necessary, be operably connected to a suitable terminator.
The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like am- picillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance. A frequently used mammalian marker is the dihydrofolate reductase gene (DHFR). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyl- transferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate. decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate . resistance markers, as- well as equivalents from other species. Particularly, for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector. To direct a subtilase variant of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al.).
More than one copy of a nucleic acid sequence encoding an enzyme of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
The nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.
Host cells
The DNA sequence encoding a subtilase variant of the present invention may be either homologous or heterologous to the host cell into which it is introduced. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to an- other promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include: a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic. sequence. The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present subtilase variants, such as prokaryotes, e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells. Examples of bacterial host cells which, on cultivation, are capable of producing the subtilase variants of the invention are gram-positive bacteria such as strains of Bacillus, e.g. strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram-negative bacteria such as Escherichia coli or Pseudomonas sp. The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
When expressing the subtilase variant in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme. When expressing the subtilase variant in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or it may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
Examples of host yeast cells include cells of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansehula, or Yarrowia. In a particular embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Other useful yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansehula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F , Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J.; and Stopani, A.O.M.; editors; 2nd edition, 1987; The Yeasts,. Rose, A>H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981). Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920.
Examples of filamentous fungal cells include filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra), in particular it may of the a cell of a species of Acremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusa um, such as F. bact di- oides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. hetero- sporum, F. negundi, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sul- phureum, F. trichothecioides or F. oxysporum, Humicola, such as H. insolens or H. lanugi- nose, Mucor, such as M. miehei, Myceliophthora, such as M. thermophilum, Neurospora, such as N. crassa, Penicillium, such as P. purpurogenum, Thielavia, such as T. terrestris, Tolypocladium, or Trichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T. reesei or T. viride, or a teleomorph or synonym thereof. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.
Examples of insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions may suitably be as described in WO 89/01029 or WO 89/01028.Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222; EP 397,485). Examples of mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621 ; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341 ; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987, Haw- ley-Nelson et al., Focus 15 (1993), 73; Ciccarone et al., Focus 15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841 - 845. Mammalian cells may be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
Methods for expression and isolation of proteins-
To express an enzyme of the present invention the above mentioned host cells transformed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth. The medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Aca- demic Press, CA, 1991).
If the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates. The enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chroma- tographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the enzyme in question. The enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activ- ity are known in the art.
The enzymes of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or ex- traction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
When an expression vector comprising a DNA sequence encoding an enzyme of the present invention is transformed/traηsfected into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme. An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell. In this context homologous impurities mean any impurity (e.g. other polypeptides than the: enzyme of the invention) which originates from the hθτ . mologous cell where the enzyme. of the invention is originally obtained from.. . . .
DETERGENT APPLICATIONS
The enzyme of the invention may be added to and thus become a component of a detergent composition. The detergent composition of the invention may for example be formulated as a hand or ma- chine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations. In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase. In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Micro- bial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
Preferred commercially available protease enzymes include Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, Ovozyme® and Kannase® (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™ FN3™ and FN4™ (Genencor International Inc.).
Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1 ,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202. Preferred commercially available lipase enzymes include Lipex®, Lipolase® and Lipolase Ul- tra® (Novozymes A/S).
Amylases: Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α- amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1 ,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.). 5
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium ox- 0 ysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as 5 those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
Commercially available cellulases include Celluzyme® and Carezyme® (Novozymes A/S), Clazinase™, ;and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation). Q
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. 5 Commercially available peroxidases include Guardzyme® (Novozymes A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a com- 0 bined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating mate- 5 rials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of .1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.
The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight. When included therein the detergent will usually contain from about 1 % to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside; alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoetha- nolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides").
The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeo- lite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethyl- enediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
The detergent may contain a bleaching system which may comprise a H202 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type. The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hy- drotropes, tarnish inhibitors, or perfumes.
It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liqour, preferably 0.05-50 mg of enzyme protein per liter of wash liqour, in particular 0.1-10 mg of enzyme protein per liter of wash liqour.
The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
MATERIALS AND METHODS
Textiles
Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland. Especially type EMPA 116 (cotton textile stained with blood, milk and ink) and EMPA 117 (polyester/cotton textile stained with blood, milk and ink).
Method for producing a subtilase variant
The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities.
The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well-known proce- dures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
EXAMPLE 1
Construction of library of Savinase variants
A library, based on Savinase positions V28, I35, T71 , I72, A73, M175 and T224 (BPN' numbering) have been synthesized. The library contains exclusively TY145-suggested alterations and covers the introduced mutations V28I,A,L; I35V,A,L; T71S; I72A,G,V; A73L,G; M175V.A; T224S.A introduced in oligopeptides, some of which are doped. Doping of nucleotide bases from a desired doping of individual amino acid residues, which is used for the example below, can be calculated as described at page 18 herein. In the attached sequence listing, the doped nucleotides below have been given the nucleo- tide symbols recommended by the WIPO Standard ST25.
The constructed oligopeptide primers are listed below. The primers are named after which positions are subject to modifications, thus 28-35-CN may have alterations in positions 28 and 35, 71-72-73-NC may have alterations in positions 71 , 72 and 73, and so forth.
28-35-CN, SEQ ID NO:7
5'-TAG ATC TGG ATG AGT GGA (50%T/50%A)(80%A/10%G/10%C)(75%T/25%C) CCC TGT ATC GAG GAC AGC (75%A/25%T)(90%A/10%G)(80%C/10%T/10%G) TTT TAG ACC AGA ACC TGT-3' 28-35-NC, SEQ ID NO:8
5'-TCC ACT CAT CCA GAT CTA-3'
(I) 71-72-73-CN, SEQ ID NO:9
5'-AAT CGA ATT GTT TAA AGC AGC (65%T/35%A)(80%A/10%C/10%G)(75%T/25%C) (90%C/10%T)G(90%T/10%A) CCC GGC CAC ATG CGT GCC-3'
(II) 71-72-73-CN, SEQ ID NO:10
5'-AAT CGA ATT GTT TAA AGC AAG (65%T/35%A)(80%A/10%C/10%G)(75%T/25%C) (90%C/10%T)G(90%T/10%A) CCC GGC CAC ATG CGT GCC-3'
(III) 71-72-73-CN, SEQ ID NO:11
5'-AAT CGA ATT GTT TAA AGC GCC (65%T/35%A)(80%A/10%C/10%G)(75%T/25%C) (90%C/10%T)G(90%T/10%A) CCC GGC CAC ATG CGT GCC-3' 71-72-73-NC, SEQ ID NO:12
5' GCT TTA AAC AAT TCG ATT 3'
139, SEQ ID NO:13
5'-GAT TAA CGC GTT GCC GCT TCT GCG-3' (I) 175-CN (90%), SEQ ID NO:14
5'-ATC AGT AGC TCC GAC TGC CA(90%T/10%C) TGC GTT CGC ATA GCG CGC-3' (II) 175-CN (10%), SEQ ID NO: 15
5'-ATC AGT AGC TCC GAC TGC CGC TGC GTT CGC ATA GCG CGC-3'
175-NC, SEQ ID NO: 16
5'-GCA GTC GGA GCT ACT GAT-3'
224-CN, SEQ ID NO: 17
5'-CGC ACC TGC AAC ATG AGG CG(80%T/10%C/10%A) AGC CAT CGA TGT ACC GI S'
224-NC, SEQ ID NO:18
5'-CCT CAT GTT GCA GGT GCG-3'
317, SEQ ID NO:19 5'-TGG CGC AAT CGG TAC CAT GGG G-3'
The Savinase gene is used as template for five individual PCR reactions under standard PCR conditions (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) where the oligos are combined as follows: 317 with 28-35-CN, 28-35-NC with 71-72-73-CN (a mixture of 80% (I) 71-72-73-CN, 10% (II) 71-72-73-CN and 10% (III) 71-72-73-CN), 71-72-73-NC with 175-CN (a mixture of 90% (l)175-CN and 10% (ll)175-CN), 175-NC with 224-CN, 224-NC with 139, giving PCR products of 125 bp, 126 bp, 3,12 bp, 165 bp and 158 bp respectively. The library is assembled by an additional PCR reaction where the five PCR products are mixed in equal molar amounts. Thereby the library contains a large number of different Savinase variants altered in one or more of the mentioned positions. The PCR reaction was assembled using a PTC-200 DNA Engine (MJ Research, Watertown, MA) and the following cycling parameters: 1 cycle of 2 min at 94 °C followed by 25 cycles of 30 sec at 94 °C, 30 sec at 55 °C and 1 min at 68 °C, and 1 cycle of 2 min at 68 °C. The library was cloned by PCR multimerization (Shafikhani et al. 1997) into Savinase expression vector psx222 and transformed into a B. subtilis host for expression. Subsequently Savinase variants can be isolated from the library, purified and characterized.
Likewise, properties from a BPN' like subtilase could be transferred to TY145 like subtilase by applying a similar procedure.
EXAMPLE 2
Transfer of regions from TY-145 to BPN' subtilases
The below mentioned highly mobile regions in TY145 have been selected for transfer from
TY145 to Savinase. The Savinase regions (BPN' numbering) are deleted and the TY145 re- gions (SEQ ID NO:1) are inserted instead. In addition regions can be selected for transfer between the psycrophiles TA41/TA39 and BPN' type protease like Savinase, or from TA39/TA41 to TY145 type non-psychrophilic subtilases. SEGMENT I
TY145 SAKDSLIASAVD, positions 144-155 (SEQ ID NO: 22)
Savinase PSPSATLEQAVN, positions 129-140 (SEQ ID NO: 23)
SEGMENT II
TY145 AGNSGSGSNTIGFPGGLV, positions 168-185 (SEQ ID NO: 24)
Savinase SGNSGAGSISYPARYA, positions 153-172 (SEQ ID NO: 25)
SEGMENT IV TY145 ASVESTWYTGGYNTIS, positions 233-248 (SEQ ID NO: 26)
Savinase VNVQSTYPGSTYASLN, positions 203-218 (SEQ ID NO: 27)
Savinase variants modified by receiving respectively segments II (Hybrid II), IV (Hybrid IV) or l+ll (Hybrid l+ll) from TY145 were observed to exhibit subtilase activity as determined by the formation of clearing zones on skim milk powder plates.
EXAMPLE 3
Transfer of regions from S39 and S41 to BPN' subtilases
The below mentioned highly mobile regions in the TA39 subtilase S39 and the TA41 subti- lase S41 , determined by the previous described homology building programs, have been selected for transfer to Savinase. The Savinase regions (BPN' numbering) are deleted and the S39 regions or S41 regions are inserted instead. Below, the S39 and S41 regions are numbered according to Figure 1. In addition regions can be selected for transfer between the psychrophiles TA41/TA39 and TY145 type non-psychrophilic subtilases. Savinase variant V104S is used as acceptor for the S39 segment II.
SEGMENT I
S39 MSLGSSG, positions 137-143 (SEQ ID NO: 28)
Savinase2 LSLGSPS, positions 124-130 (SEQ ID NO: 29)
SEGMENT II
S39 MSLGSSGESSLI, positions 137-148 (SEQ ID NO: 30)
Savinase variant V104S LSLGSPSPSATL, positions 124-135 (SEQ ID NO: 31)
SEGMENT III
S39 NNSSITQT, positions 15-22 (SEQ ID NO: 32)
Savinase VQAPAAHN, positions 11-18 (SEQ ID NO: 33) SEGMENT IV
S39 GTTYTN, positions 55-62 (SEQ ID NO: 34)
Savinase2 VPG*EPST, positions 51*-58 (SEQ ID NO:35)
SEGMENT V
S39 RQ, positions 68-69
Savinase GN, positions 61-62
SEGMENT VI S39 SGESSLI, positions 142-148 (SEQ ID NO: 36)
Savinase PSPSATL, positions 129-135 (SEQ ID NO: 37)
SEGMENT VII
S39 WFDGGYATI, positions 237-245 (SEQ ID NO: 38) Savinase YPGSTYASL, positions 209-217 (SEQ ID NO: 39)
Savinase variants modified by receiving respectively segments I or II from S39 were observed to have subtilase activity against the substrate suc-AAPF-pNA (Suc-Ala-Ala-Pro-Phe- pNA). The subtilase activity was determined in a temperature profile assay where specific activities i.e. micromole substrate per minute per mg enzyme against before mentioned substrates, were determined at every 5 degrees Celsius. The measurements were done in a Tris-base buffer pH 9.
To measure subtilase activity in suc-AAPF-pNA: 100 uL 1.56 mM Suc-Ala-Ala-Pro-Phe-pNA in 0.1 M Tris was added to 100 uL Tris-base, pH 9.0 buffer and 20 uL enzyme. The devel- opment of the degradation product pNA (paranitrophenol) was measured as initial velocities at 405 nm on an Elisa Reader for 1 minute.
The Savinase variant with segment I substituted had less specific activity against suc-AAPF- pNA compared to Savinase, whereas the Savinase variant with segment II substituted had more than 2 times higher specific activity against suc-AAPF-pNA than Savinase. In an AMSA-test (performed like described in Example 5 herein) the wash performance was shown to be preserved in Savinase variant with segment II compared to Savinase.
Further, four Savinase variants were constructed with the following combinations of seg- ments from S39:
Segments III, V and VII; Segments III and V; Segments III, V, VI and Segments III and IV. All four Savinase variants showed subtilase activity on skim milk plates. Segments from the S41 subtilase suggested for transfer to Savinase are:
SEGMENT VIII S41 GTNFTD, positions 55-62 (SEQ ID NO: 40) Savinase VPG*EPST, positions 51-58 (SEQ ID NO: 41)
SEGMENT IX
S41 NGGTGS, positions 82-87 (SEQ ID NO: 42)
Savinase ALNNSI, positions 74-79 (SEQ ID NO: 43)
SEGMENT X
S41 DDGSGYA, positions 106-112 (SEQ ID NO: 44)
Savinase ASGSGSV, positions 98-104 (SEQ ID NO: 45)
SEGMENT XI S41 WAQSPAA, positions 263-269 (SEQ ID NO: 46) Savinase KQKNPSW, positions 235-241 (SEQ ID NO: 47)
Four Savinase variants were constructed with the following segments from S41 : Segment X; Segments IX and X; Segments VIII and X; and Segments X and XI. All four Savinase variants showed subtilase activity on skim milk plates.
AMSA wash tests were performed on variants with Segment X and Segments X and XI like described in Example 5 herein. The assay was conducted under the experimental conditions specified below:
Figure imgf000046_0001
Figure imgf000047_0001
The wash performance score (described in Example 5 herein) of the Savinase variants with Segment X and Segments X and XI was S (1) indicating an improved wash performance compared to Savinase.
EXAMPLE 4
Purification and assessment of enzyme concentration
After fermentation, purification of subtilisin variants is accomplished using Hydrophobic Charge Induction Chromatography (HCIC) and subsequent vacuum filtration. To capture the enzyme, the HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl-Pyridine (4-MEP) is bound.
Beads of the cellulose matrix sized 80-100 μm are mixed with a media containing yeast and the transformed B. subtilis capable of secreting the subtilisin variants and incubated at pH 9.5 in Unifilter® microplates.
As 4-MEP is hydrophobic at pH > 7 and the subtilisin variants are hydrophobic at pH 9.5 a hydrophobic association is made between the secreted enzyme and the 4-MEP on the beads. After incubation the media and cell debris is removed by vacuum filtration while the beads and enzyme are kept on the filter. To elute the enzyme from the beads the pH is now lowered by washing the filter with an elu- tion buffer (pH 5). Hereby the enzymes part from the beads and can be retrieved from the buffer.
The concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST).
The purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites. The protease and inhibitor binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive. To measure the residual protease activity, a substrate suc-AAPF-pNA (0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer) is added after the incubation with inhibitor and during the following 4 minutes the development of the degradation product pNA (paranitrophenol) is measured periodically at 405 nm on an Elisa Reader. EXAMPLE 5
Wash performance of detergent compositions comprising modified enzymes Wash performance of detergent compositions comprising enzyme hybrids or enzyme variants of the present is tested at low washing temperature.
The Savinase variant Hybrid IV of Example 2 was tested for washing performance in two different assays; a microlitre scale assay (AMSA) and a millilitre scale assay (Mini wash).
AMSA The enzyme variants of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA test the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress. For further description see WO 02/42740 especially the paragraph "Special method embodiments" at page 23-24.
The assay was conducted under the experimental conditions specified below:
Figure imgf000048_0001
After washing the textile pieces were flushed in tap water and air-dried.
The performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific enzyme variant. Brightness can also be expressed as the intensity of the light reflected from the textile sample when luminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a clean textile. Therefore the intensity of the reflected light can be used to measure wash performance of an enzyme variant.
Colour measurements are made with a professional flatbed scanner (PFU DL2400prό), which is used to capture an image of the washed textile samples. The scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits. In order to get accurate results, the scanner is frequently calibrated with a Kodak reflective IT8 target.
To extract a value for the light intensity from the scanned images, a special designed software application is used (Novozymes Color Vector Analyzer). The program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
Figure imgf000049_0001
The wash performance (P) of the variants was calculated in accordance with the below formula: P lnt(v) - lnt(r) where
Int(v) is the light intensity value of textile surface washed with enzyme variant and lnt(r) is the light intensity value of textile surface washed with the reference enzyme subtilisin
309 (BLSAVl).
The result of the AMSA wash of Hybrid IV was a Performance Score of S (2) in accordance with the definition:
Performance Scores (S) are summing up the performances (P) of the tested enzyme variants as: S (2) which indicates that the variant performs better than the reference at all three concentrations (5, 10 and 30 nM) and
S (1) which indicates that the variant performs better than the reference at one or two concentrations. Mini wash assay
The millilitre scale wash performance assay was conducted under the following conditions:
Figure imgf000050_0001
After wash the measurement of remission from the test material was done at 460 nm using a Zeiss MCS 521 VIS spectrophotometer. The measurements were done according to the manufacturer's protocol.
As shown in Table 1 the textile washed with the Savinase variant Hybrid IV at 20°C in Omo Acao has a higher remission than the textile washed with the parent. This result indicates that this variant has better wash performance at low temperature than the parent Savinase.
Table 1. Wash performance results of the subtilase variant in Omo Acao for a dosage of 5 nM and 10 nM enzyme.
Figure imgf000050_0002
As it can be concluded from Table 1 the modified subtilases of the invention exhibits an improvement in wash performance. EXAMPLE 6
Wash performance of detergent compositions comprising enzyme variants of the present invention was tested at low washing temperature using the Automatic Mechanical Stress Assay (AMSA) as described in Example 5 herein.
Table 2. AMSA wash results of subtilase variants.
Figure imgf000051_0001
APPENDIX 1
REMARK REFINEMENT . REMARK PROGRAM REFMAC 5.0 REMARK AUTHORS MURSHUDOV, VAGIN, DODSON REMARK REMARK REFINEMENT TARGET : MAXIMUM LIKELIHOOD REMARK REMARK DATA USED IN REFINEMENT. REMARK RESOLUTION RANGE HIGH (ANGSTROMS) 1.80 REMARK RESOLUTION RANGE LOW (ANGSTROMS) 56.80 REMARK DATA CUTOFF (SIGMA (F) ) NONE REMARK COMPLETENESS FOR RANGE (%) 99.88 REMARK NUMBER OF REFLECTIONS 38045 REMARK REMARK FIT TO DATA USED IN REFINEMENT. REMARK CROSS-VALIDATION METHOD THROUGHOUT REMARK FREE R VALUE TEST SET SELECTION RANDOM REMARK R VALUE (WORKING + TEST SET) 0.15648 REMARK R VALUE (WORKING SET) 0.15487 REMARK FREE R VALUE 0.18707 REMARK FREE R VALUE TEST SET SIZE (%) 5.0 REMARK FREE R VALUE TEST SET COUNT 2009 REMARK REMARK FIT IN THE HIGHEST RESOLUTION BIN. REMARK TOTAL NUMBER OF BINS USED 20 REMARK BIN RESOLUTION RANGE HIGH 1.796 REMARK BIN RESOLUTION RANGE LOW 1.842 REMARK REFLECTION IN BIN (WORKING SET) 2738 REMARK BIN R VALUE (WORKING SET) 0.191 REMARK BIN FREE R VALUE SET COUNT 138 REMARK BIN FREE R VALUE 0.234 REMARK REMARK NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK ALL ATOMS : 3156 REMARK REMARK B VALUES. REMARK FROM WILSON PLOT (A**2) NULL REMARK MEAN B VALUE (OVERALL, A**2) 14.804 REMARK OVERALL ANISOTROPIC B VALUE. REMARK Bll (A**2) 0 28 REMARK B22 (A**2) -0 86 REMARK B33 (A**2) 0 58 REMARK B12 (A**2) 0 00 REMARK B13 (A**2) 0 00 REMARK B23 (A**2) 0 00 REMARK REMARK ESTIMATED OVERALL COORDINATE ERROR. REMARK ESU BASED ON R VALUE (A) : 0.100 REMARK ESU BASED ON FREE R VALUE (A) : 0.098 REMARK ESU BASED ON MAXIMUM LIKELIHOOD (A) : 0.093 REMARK ESU FOR B VALUES BASED ON MAXIMUM LIKELI IHHOOOODD (A**2) : 2.910 REMARK REMARK CORRELATION COEFFICIENTS. REMARK CORRELATION COEFFICIENT FO-FC 0.963 REMARK CORRELATION COEFFICIENT FO-FC FREE 0.952 REMARK REMARK RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK BOND LENGTHS REFINED ATOMS (A) 2798 ; 0.021 • 0.021 REMARK BOND LENGTHS OTHERS (A) 2500 ; 0.001 • 0.020 REMARK BOND ANGLES REFINED ATOMS (DEGREES) 3805 ; 1.859 • 1.943 REMARK BOND ANGLES OTHERS (DEGREES) 5821 ; 0.854 • 3.000 REMARK 3 TORSION ANGLES, PERIOD 1 (DEGREES) 372 5.125 3.000
REMARK 3 TORSION ANGLES, PERIOD 3 (DEGREES) 462 16 .877 15.000
REMARK 3 CHIRAL-CENTER RESTRAINTS (A* *3) 437 0 .119 0.200
REMARK 3 GENERAL PLANES REFINED ATOMS (A) 3201 0 .009 0.020
REMARK 3 GENERAL PLANES OTHERS (A) 535 0 .004 0.020
REMARK 3 NON-BONDED CONTACTS REFINED ATOMS (A) 610 0 .228 0.300
REMARK 3 NON-BONDED CONTACTS OTHERS (A) 2548 0 .203 0.300
REMARK 3 H-BOND (X...Y) REFINED ATOMS (A) 374 0 .184 0.500
REMARK 3 H-BOND (X...Y) OTHERS (A) 3 0 .279 0.500
REMARK 3 POTENTIAL METAL-ION REFINED ATOMS (A) 16 0 .119 0.500
REMARK 3 SYMMETRY VDW REFINED ATOMS (A) 7 0 .127 0.300
REMARK 3 SYMMETRY VDW OTHERS (A) 27 0 .152 0.300
REMARK 3 SYMMETRY H-BOND REFINED ATOMS (A) 37 0 .278 0.500
REMARK 3
REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS COUNT RMS WEIGHT
REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A* *2) 1840 1 .131 1.500
REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A* *2) 2941 1 .781 2.000
REMARK 3 SIDE-CHAIN BOND REFINED ATOMS (A* *2) 958 2 .873 3.000
REMARK 3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2) 864 • 4 .300 4.500
REMARK 3
REMARK 3 NCS RESTRAINTS STATISTICS
REMARK 3 NUMBER OF NCS GROUPS : NULL
REMARK 3
REMARK 3
REMARK 3 TLS DETAILS
REMARK 3 NUMBER OF TLS GROUPS : NULL
REMARK 3
REMARK 3
REMARK 3 BULK SOLVENT MODELLING.
REMARK 3 METHOD USED : BABINET MODEL WITH MASK
REMARK 3 PARAMETERS FOR MASK CALCULATION
REMARK 3 VDW PROBE RADIUS 1.40
REMARK 3 ION PROBE RADIUS : 0.80
REMARK 3 SHRINKAGE RADIUS : 0.80
REMARK 3
REMARK 3 OTHER REFINEMENT REMARKS:
REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
REMARK 3
CISPEP 1 GLY A 172 SER A 173 0.00
CISPEP 2 PHE A 180 PRO A 181 0.00
SSBOND 1 CYS A 52 CYS A 66
CRYST1 58 753 66.838 107.082 90.00 90.00 90.00 P 21 21 21
SCALE1' 0.017020 0.000000 0.000000 0. D0000
SCALE2 0.000000 0.014962 0.000000 0. .0000
SCALE3 0.000000 0.000000 0.009339 0. -0000
ATOM 1 N ALA A 1 2.336 20.870 1 .027 1. DO 27.48 N
ATOM 3 CA ALA A 1 1.951 20.940 2 .465 1. -0 29.42 C
ATOM 5 CB ALA A 1 2.391 19.637 3 .197 1. DO 29.45 C
ATOM 9 C ALA A 1 2.665 22.149 3 .096 1. DO 28.87 C
ATOM 10 O ALA A 1 3.696 22.577 2 .627 1. DO 30.49 O
ATOM 13 N VAL A 2 2.014 22.747 4 .052 1. DO 30.31 N
ATOM 15 CA VAL A 2 2.658 23.754 4 .877 1. DO 30.69 C
ATOM 17 CB VAL A 2 2.068 25.139 4 .604 1. DO 30.94 C
ATOM 19 CGI VAL A 2 2.611 25.702 3 .252 1. DO 32.62 C
ATOM 23 CG2 VAL A 2 0.577 25.086 4 .667 1. DO 30.84 C
ATOM 27 C VAL A 2 2.494 23.346 6 .347 1. DO 29.48 C
ATOM 28 O VAL A 2 1.580 22.610 6 .743 1. DO 29.33 O
ATOM 29 N PRO A 3 3.412 23.788 7 .186 1. DO 28.10 N
ATOM 30 CA PRO A 3 3.298 23.380 8 .581 1. DO 27.90 C
ATOM 32 CB PRO A 3 4.645 23.830 9 .185 1. DO 26.48 C
ATOM 35 CG PRO A 3 5.116 24.998 8 .340 1. DO 26.57 C
ATOM 38 CD PRO A 3 4.530 24.697 6 .933 1. DO 27.60 C
ATOM 41 C PRO A 3 2.129 24.112 9 .216 1. DO 27.01 C ATOM 42 O PRO A 3 1.602 25.037 8.600 .00 28.48 O
ATOM 43 N SER A 4 1.767 23.774 10.434 .00 26.37 N
ATOM 45 CA SER A 4 0.718 24.505 11.159 .00 25.29 C
ATOM 47 CB SER A 4 0.279 23.780 12.444 .00 26.52 C
ATOM 50 OG SER A 4 1.173 23.913 13.554 .00 25.41 O
ATOM 52 C SER A 4 1.105 25.956 11.445 .00 26.09 C
ATOM 53 O SER A 4 0.212 26.810 11.603 .00 25.32 O
ATOM 54 N THR A 5 2.415 26.233 11.497 .00 24.12 N
ATOM 56 CA THR A 5 3.023 27.567 11.741 .00 22.81 C
ATOM 58 CB THR A 5 3.009 28.007 13.237 .00 24.21 C
ATOM 60 OGl THR A 5 3.793 29.191 13.386 1.00 23.29 O
ATOM 62 CG2 THR A 5 3.727 26.999 14.151 .00 22.71 c
ATOM 66 C THR A 5 4.413 27.436 11.219 .00 23.10 c
ATOM 67 O THR A 5 5, .012 26.322 11.267 .00 21.48 O
ATOM 68 N GLN A 6 4 .,959 28.518 10.692 .00 21.30 N
ATOM 70 CA GLN A 6 6 .,260 28.438 10.102 .00 21.50 C
ATOM 72 CB GLN A 6 6 ..512 29.634 9..209 .00 22.77 C
ATOM 75 CG GLN A 6 5 .,626 29.570 7..926 .00 23.15 C
ATOM 78 CD GLN A 6 5 .999 30.579 6..911 .00 28.40 C
ATOM 79 OE1 GLN A 6 5 .356 31.619 6.822 .00 30.34 O
ATOM 80 NE2 GLN A 6 7 ..016 30.300 6.133 1.00 24.59 N
ATOM 83 C GLN A 6 7.295 28.389 11.185 1.00 20.24 C
ATOM 84 O GLN A 6 8.438 28.017 10.927 .00 18.46 O
ATOM 85 N THR A 7 6.870 28.777 12.378 .00 18.84 N
ATOM 87 CA THR A 7 7.752 28.808 13.565 .00 19.27 C
ATOM 89 CB THR A 7 8.135 30.238 13.914 .00 19.55 C
ATOM 91 OGl THR A 7 6.958 31.041 14.091 1.00 23.00 O
ATOM 93 CG2 THR A 7 8.910 30.878 12.842 1.00 19.61 C
ATOM 97 C THR A 7 7 .111 28.128 14.755 .00 18.28 C
ATOM 98 O THR A 7 6 .436 28.735 15.547 .00 18.57 o
ATOM 99 N PRO A 8 7 .288 26.803 14.834 .00 17.34 N
ATOM 100 CA PRO A 8 6 .795 26.000 15.922 .00 17.79 C
ATOM 102 CB PRO A 8 7 .459 24.615 15.659 .00 17.18 C
ATOM 105 CG PRO A 8 7 .556 24.570 14.138 .00 18.32 C
ATOM 108 CD PRO A 8 7 ..961 25.984 13.814 .00 16.59 C
ATOM 111 C PRO A 8 7 .162 26.584 17.273 .00 16.99 C
ATOM 112 O PRO A 8 8 .105 27.339 17.369 .00 18.02 O
ATOM 113 N TRP A 9 6 .426 26.203 18.280 .00 18.21 N
ATOM 115 CA TRP A 9 6. .613 26.750 19.611 .00 17.56 C
ATOM 117 CB TRP A 9 5 .723 26.059 20.603 1.00 17.84 C
ATOM 120 CG TRP A 9 6 .129 24.806 21.197 1. 00 14.36 C
ATOM 121 CD1 TRP A 9 5 .,772 23.569 20.796 1.00 15.37 C
ATOM 123 NE1 TRP A 9 6.278 22.630 21.658 1, 00 15.66 N
ATOM 125 CE2 TRP A 9 7.033 23.276 22.609 1.00 16.83 C
ATOM 126 CD2 TRP A 9 6.952 24.642 22.345 1.00 14.81 C
ATOM 127 CΞ3 TRP A 9 7.642 25.531 23.186 1, 00 14.62 C
ATOM 129 CZ3 TRP A 9 8.362 24.982 24.301 1, 00 11.92 C
ATOM 131 CH2 TRP A 9 8.393 23.626 24.526 1, 00 15.97 C
ATOM 133 CZ2 TRP A 9 7.757 22.750 23.677 1, 00 14.67 C
ATOM 135 C TRP A 9 8.073 26.760 20.083 1, 00 18.17 C
ATOM 136 O TRP A 9 8.531 27.737 20.662 1 00 15.91 O
ATOM 137 N GLY A 10 8.780 25.675 19.859 1 00 16.36 N
ATOM 139 CA GLY A 10 10.180 25.618 20.307 1 00 15.48 C
ATOM 142 C GLY A 10 11.108 26.619 19.663 1 00 15.80 C
ATOM 143 O GLY A 10 12.089 27.060 20.254 1 00 14.71 O
ATOM 144 N ILE A 11 10.801 26.939 18.410 1.00 15.18 N
ATOM 146 CA ILE A 11 11.599 27.866 17.642 1, 00 15.64 C
ATOM 148 CB ILE A 11 11.303 27.768 16.151 1, 00 14.65 C
ATOM 150 CGI ILE A 11 11.479 26.328 15.653 1.00 15.91 C
ATOM 153 GDI ILE A 11 12.945 25.811 15.725 1 00 16.96 C
ATOM 157 CG2 ILE A 11 12.204 28.704 15.385 1 00 16.09 C
ATOM 161 C ILE A 11 11.291 29.225 18.193 1 00 15.73 C
ATOM 162 O ILE A 11 12.197 29.995 18.438 1 00 15.02 O ATOM 163 N LYS A 12 10.,005 29,.552 18..352 1..00 16..62 N
ATOM 165 CA LYS A 12 9. ,649 30, .832 18. .949 1. ,00 16. .28 C
ATOM 167 CB LYS A 12 8. ,147 30, .926 19. .078 1. ,00 17. .21 C
ATOM 170 CG LYS A 12 7. ,419 31, .148 17. .707 1. ,00 20. .12 C
ATOM 173 CD LYS A 12 5, .874 31 .303 17, .997 1, .00 20. .50 C
ATOM 176 CE LYS A 12 5. ,137 31, .855 16. .795 1. .00 28. .93 C
ATOM 179 NZ LYS A 12 4. .564 30, .819 15. .858 1. ,00 19. .42 N
ATOM 183 C LYS A 12 10. .242 30, .956 20. .345 1. .00 15. .82 C
ATOM 184 O LYS A 12 10, .790 32 .014 20. .745 1. .00 15, .87 O
ATOM 185 N SER A 13 10, .172 29 .865 21, .075 1, .00 13, .09 N
ATOM 187 CA SER A 13 10, .655 29 .893 22, .450 1. .00 13. .64 C
ATOM 189 CB SER A 13 10. .284 28, .586 23. .149 1. .00 12. .46 C
ATOM 192 OG SER A 13 10. .790 28, .548 24. .491 1. ,00 14. .12 O
ATOM 194 C SER A 13 12, .167 30 .131 22. .519 1. .00 12. .81 C
ATOM 195 O SER A 13 12, .650 30 .959 23, .323 1, .00 11, .51 O
ATOM 196 N ILE A 14 12, .931 29 .371 21, .752 1, .00 12. .90 N
ATOM 198 CA ILE A 14 14, .391 29 .521 21, .839 1. .00 13 .05 C
ATOM 200 CB ILE A 14 15. .108 28, .318 21. .206 1. .00 12, .73 C
ATOM 202 CGI ILE A 14 16, .498 28 .183 21, .810 1, .00 13 .73 C
ATOM 205 GDI ILE A 14 17, .265 26 .959 21, .415 1, .00 17 .32 C
ATOM 209 CG2 ILE A 14 15, .161 28 .394 19, .753 1, .00 14. .13 C
ATOM 213 C ILE A 14 14, .869 30 .861 21, .299 1, .00 14 .41 C
ATOM 214 O ILE A 14 15. .907 31 .367 21. .680 1, .00 15 .23 O
ATOM 215 N TYR A 15 14, .094 31 .423 20, .389 1, .00 15 .25 N
ATOM 217 CA TYR A 15 14, .392 32 .753 19, .877 1. .00 16, .87 C
ATOM 219 CB TYR A 15 13, .742 32 .965 18, .490 1. .00 15, .13 C
ATOM 222 CG TYR A 15 14, .683 32 .629 17, .348 1, .00 16, .40 C
ATOM 223 CD1 TYR A 15 14 .956 31 .303 17 .008 1 .00 14 .01 C
ATOM 225 CE1 TYR A 15 15, .834 30 .998 16, .024 1, .00 16, .36 C
ATOM 227 CZ TYR A 15 16, .453 31 .993 15, .321 1. .00 16, .82 C
ATOM 228 OH TYR A 15 17, .364 31, .738 14, .329 1. .00 15, .20 O
ATOM 230 CΞ2 TYR A 15 16, .182 33 .303 15 .621 1, .00 17 .27 C
ATOM 232 CD2 TYR A 15 15, .320 33 .610 16 .628 1, .00 16 .64 C
ATOM 234 C TYR A 15 13, .925 33 .826 20 .856 1, .00 16 .27 C
ATOM 235 O TYR A 15 14. .311 35 .008 20, .744 1. .00 17, .84 O
ATOM 236 N ASN A 16 13. .075 33 .432 21, .780 1. .00 17, .21 N
ATOM 238 CA ASN A 16 12. .534 34 .334 22, .811 1. .00 17 .53 C
ATOM 240 CB ASN A 16 13, .628 34 .860 23, .743 1, .00 16 .83 C
ATOM 243 CG ASN A 16 13. .098 35 .265 25, .103 1. .00 18 .27 c
ATOM 244 OD1 ASN A 16 11, .901 35 .461 25 .288 1. .00 21 .90 o
ATOM 245 ND2 ASN A 16 13. .987 35, .324 26, .075 1. .00 17, .31 N
ATOM 248 C ASN A 16 11. .788 35 .480 22. .114 1. .00 19 .04 c
ATOM 249 O ASN A 16 11, .940 36 .642 22, .463 1. .00 18 .19 o
ATOM 250 N ASP A 17 10, .972 35 .107 21, .135 1, .00 18 .56 N
ATOM 252 CA ASP A 17 10, .176 36 .069 20, .372 1, .00 19 .69 C
ATOM 254 CB ASP A 17 11 .019 36 .634 19 .287 1 .00 19 .45 C
ATOM 257 CG ASP A 17 10, .362 37 .812 18, .579 1, .00 20 .65 C
ATOM 258 OD1 ASP A 17 9, .160 38 .017 18, .745 1, .00 24 .61 0
ATOM 259 OD2 ASP A 17 11, .034 38 .547 17, .849 1, .00 19 .73 0
ATOM 260 C ASP A 17 8, .937 35 .412 19, .778 1, .00 19 .80 C
ATOM 261 O ASP A 17 9 .032 34 .693 18 .834 1. .00 22 .05 0
ATOM 262 N GLN A 18 7 .791 35 .647 20 .369 1 .00 20 .51 N
ATOM 264 CA GLN A 18 6, .593 34 .957 19, .960 1, .00 21 .49 C
ATOM 266 CB GLN A 18 5, .549 35 .062 21, .054 1, .00 22 .51 C
ATOM 269 CG GLN A 18 5 .917 34 .348 22. .318 1. .00 24 .55 C
ATOM 272 CD GLN A 18 6 .243 32 .907 22 .041 1 .00 29 .84 C
ATOM 273 OE1 GLN A 18 7 .347 32 .450 22 .314 1 .00 31 .44 O
ATOM 274 NE2 GLN A 18 5, .301 32 .204 21, .457 1, .00 28, .00 N
ATOM 277 C GLN A 18 6, .076 35 .511 18, .658 1, .00 21 .92 C
ATOM 278 O GLN A 18 5 .213 34 .911 18 .021 1 .00 22 .98 O
ATOM 279 N SER A 19 6 .697 36 .572 18 .185 1 .00 22 .89 N
ATOM 281 CA SER A 19 6 .231 37 .202 16 .951 1 .00 23 .92 C
ATOM 283 CB SER A 19 6 .338 38 .715 17 .096 1 .00 23 .69 C ATOM 286 OG SER A 19 7.627 39.225 16.746 . 00 25 . 66 o
ATOM 288 C SER A 19 6.947 36.728 15.670 . 00 24 . 30 c
ATOM 289 O SER A 19 6.454 36.972 14.566 . 00 25 . 69 o
ATOM 290 N ILE A 20 8.079 36.029 15.764 . 00 23 . 62 N
ATOM 292 CA ILE A 20 8.791 35.673 14.529 . 00 23 . 92 C
ATOM 294 CB ILE A 20 10.104 34.965 14.836 . 00 24 . 53 c
ATOM 296 CGI ILE A 20 9.858 33.755 15.727 . 00 22 .48 c
ATOM 299 CD1 ILE A 20 11.041 32.826 15.641 . 00 23 . 83 c
ATOM 303 CG2 ILE A 20 11.127 35.902 15.477 . 00 27 .13 c
ATOM 307 C ILE A 20 8.011 34.784 13.573 . 00 23 .40 c
ATOM 308 O ILE A 20 7.241 33.913 13.999 . 00 24 . 30 o
ATOM 309 N THR A 21 8.295 34.963 12.296 . 00 23 . 97 N
ATOM 311 CA THR A 21 .686 34.184 11.215 1, .00 24 .95 C
ATOM 313 CB THR A 21 .856 35.107 10.283 1 .00 24 .97 C
ATOM 315 OGl THR A 21 .690 36.186 9.842 1. .00 27 .61 O
ATOM 317 CG2 THR A 21 .771 35.794 11.039 1, .00 28 .02 C
ATOM 321 C THR A 21 8.799 33.570 10.392 1, .00 23 .92 C
ATOM 322 O THR A 21 8.544 32.865 9.419 1, .00 23 .90 o
ATOM 323 N LYS A 22 10.044 33.862 10.735 . 00 23 . 93 N
ATOM 325 CA LYS A 22 11.148 33.239 10.041 . 00 24 . 18 C
ATOM 327 CB LYS A 22 11.386 33.880 8.675 . 00 25 . 22 C
ATOM 330 CG LYS A 22 11.830 35.314 8.724 . 00 28 . 95 C
ATOM 333 CD LYS A 22 12.397 35.808 7.348 . 00 32 . 55 C
ATOM 336 CE LYS A 22 13.701 35.129 0. .10 31 .56 C
ATOM 339 NZ LYS A 22 14.274 35.671 5.722 0. .10 31 .66 N
ATOM 343 C LYS A 22 12.406 33.333 10.903 1. .00 23 .63 C
ATOM 344 O LYS A 22 12.471 34.145 11.823 1. .00 24 .17 O
ATOM 345 N THR A 23 13.395 32.491 10.628 1. .00 21 .41 N
ATOM 347 CA THR A 23 14.661 32.526 11.342 1. .00 20 .21 C
ATOM 349 CB THR A 23 14.914 31.202 12.030 1. 00 20 .28 C
ATOM 351 OGl THR A 23 14.859 30.158 11.034 1. 00 19 .28 O
ATOM 353 CG2 THR A 23 13.846 30.915 13.074 1. .00 21 .44 C
ATOM 357 C THR A 23 15.785 32.791 10.384 1. .00 20 .28 C
ATOM 358 O THR A 23 15.565 32.740 9.182 1. ,00 19 .80 O
ATOM 359 N THR A 24 16.996 33.027 10.908 1. 00 19 .65 N
ATOM 361 CA THR A 24 18.201 33.246 10.130 1. 00 19 .91 C
ATOM 363 CB THR A 24 18.532 34.784 9.840 1 1. . ,0000 2 211 . .9933 C
ATOM 365 OGl THR A 24 18.685 35.435 11.102 1 1. . ,0000 2 255 . .6699 O
ATOM 367 CG2 THR A 24 17.402 35.514 9.229 1 1. . ,0000 2 255 . .0022 C
ATOM 371 C THR A 24 19.407 32.743 10.928 1 1. . ,0000 1 199 . .5555 C
ATOM 372 O THR A 24 19.372 32.551 12.149 1 1. . 0000 1 199,, . .7700 O
ATOM 373 N GLY A 25 20.473 32.497 10.225 1 1. . ,0000 1 177 . .4411 N
ATOM 375 CA GLY A 25 21.716 32.187 10.893 1 1. . ,0000 1 177 . .6644 C
ATOM 378 C GLY A 25 22.286 30.799 10.641 1 1. . ,0000 1 166 . .1166 C
ATOM 379 O GLY A 25 21.583 29.936 10.124 1 1. . ,0000 1 155 . .9922 O
ATOM 380 N GLY A 26 23.548 30.620 11.068 1 1. . ,0000 1 155 . .4499 N
ATOM 382 CA GLY A 26 24.296 29.380 10.938 1 1. . ,0000 1 155 . .4444 C
ATOM 385 C GLY A 26 25.166 29.220 9.692 1 1. . ,0000 1 155 . .6611 C
ATOM 386 O GLY A 26 25.782 28.199 9.493 1 1. . ,0000 1 155 . .3366 O
ATOM 387 N SER A 27 25.264 30.249 8.861 1 1. . ,0000 1 177 . .7722 N
ATOM 389 CA SER A 27 26.108 30.182 7.691 1 1. . ,0000 1 188 . .4455 C
ATOM 391 CB SER A 27 25.990 31.500 6.837 1 1. . ,0000 2 200 . .0088 C
ATOM 394 OG SER A 27 26.686 32.496 7.490 1 1. . ,0000 2 266 . .3344 O
ATOM 396 C SER A 27 27.534 29.838 8.029 1 1. . ,0000 1 177 . .4455 C
ATOM 397 O SER A 27 28.166 30.341 8.969 1 .00 17 .59 O
ATOM 398 N GLY A 28 28.071 28.913 7.241 1 .00 16 .01 N
ATOM 400 CA GLY A 28 29.421 28.494 7.385 1 .00 16 .46 C
ATOM 403 C GLY A 28 29.615 27.360 8.377 1 .00 15 .36 C
ATOM 404 O GLY A 28 30.739 26.917 8.527 1 .00 16 .82 O
ATOM 405 N ILE A 29 28.587 26.931 9.076 1 .00 14 .25 N
ATOM 407 CA ILE A 29 28.782 25.832 10.044 1 .00 13 .92 C
ATOM 409 CB ILE A 29 28.057 26.170 11.335 1 .00 14 .10 C
ATOM 411 CGI ILE A 29 28.482 27.563 11.856 1 .00 13 .61 C ATOM 414 CDl ILE A 29 29,.986 27.710 12..081 1,.00 15..86 c
ATOM 418 CG2 ILE A 29 28, .269 25, .076 12. .417 1, .00 14. .40 c
ATOM 422 C ILE A 29 28, .143 24 .586 9. .459 1, .00 14. .30 c
ATOM 423 O ILE A 29 27. .190 24, .708 8. .690 1. .00 14. ,55 0
ATOM 424 N LYS A 30 28 .614 23 .402 9, .853 1, .00 13. .73 N
ATOM 426 CA LYS A 30 28 .008 22 .173 9. .422 1, .00 13 .73 C
ATOM 428 CB LYS A 30 29 .019 21 .204 8, .860 1, .00 14 .64 C
ATOM 431 CG LYS A 30 30 .072 21 .822 7, .951 1, .00 15 .03 C
ATOM 434 CD LYS A 30 29 .438 22 .566 6. .745 1. .00 16 .42 C
ATOM 437 CE LYS A 30 30 .497 23 .364 5. .987 1. .00 19 .44 C
ATOM 440 NZ LYS A 30 29 .865 24 .009 4. .752 1, .00 17 .01 N
ATOM 444 C LYS A 30 27 .354 21 .482 10, .655 1, .00 13 .57 C
ATOM 445 O LYS A 30 27 .978 21 .473 11, .716 1, .00 14, .48 O
ATOM 446 N VAL A 31 26 .163 20 .941 10 .498 1 .00 13 .11 N
ATOM 448 CA VAL A 31 25 .572 20 .118 11, .583 1, .00 13 .65 C
ATOM 450 CB VAL A 31 24 .249 20 .639 12. .061 1, .00 13 .16 C
ATOM 452 CGI VAL A 31 23 .726 19 .743 13, .173 1, .00 15. .78 C
ATOM 456 CG2 VAL A 31 24 .401 22 .058 12, .573 1. .00 12 .55 C
ATOM 460 C VAL A 31 25 .470 18 .708 11 .047 1 .00 14 .30 C
ATOM 461 O VAL A 31 24 .828 18 .458 10, .003 1. .00 14 .74 O
ATOM 462 N ALA A 32 26 .129 17 .789 11. .737 1, .00 14, .62 N
ATOM 464 CA ALA A 32 26. .092 16 .387 11. .366 1, .00 13 .46 C
ATOM 466 CB ALA A 32 27 .419 15 .728 11. .606 1 .00 11 .29 C
ATOM 470 C ALA A 32 24 .972 15 .696 12. .149 1 .00 13 .08 C
ATOM 471 O ALA A 32 25, .056 15, .556 13. .393 1, .00 12, .55 O
ATOM 472 N VAL A 33 23, .916 15, .340 11. .435 1, .00 10, .76 N
ATOM 474 CA VAL A 33 22, .778 14, .654 12. .038 1. .00 11, .75 c
ATOM 476 CB VAL A 33 21 .468 15 .152 11, .453 1 .00 11 .00 c
ATOM 478 CGI VAL A 33 20 .317 14 .313 11, .935 1, .00 13 .80 c
ATOM 482 CG2 VAL A 33 21, .268 16, .618 11. .738 1. .00 12, .19 c
ATOM 486 C VAL A 33 22, .959 13, .155 11. .847 1, .00 12, .72 c
ATOM 487 O VAL A 33 22 .830 12 .623 10, .715 1 .00 11 .99 0
ATOM 488 N LEU A 34 23 .290 12 .466 12, .932 1 .00 12 .34 N
ATOM 490 CA LEU A 34 23 .599 11 .022 12, .949 1, .00 11 .72 C
ATOM 492 CB LEU A 34 24, .811 10, .744 13. .878 1, .00 11, .57 C
ATOM 495 CG LEU A 34 26, .190 10, .819 13. .262 1. .00 11, .59 C
ATOM 497 CDl LEU A 34 26 .515 12 .228 12, .621 1, .00 10 .38 C
ATOM 501 CD2 LEU A 34 27 .275 10 .414 14, .265 1, .00 12 .17 C
ATOM 505 C LEU A 34 22 .303 10 .366 13, .416 1, .00 12 .33 C
ATOM 506 O LEU A 34 21 .964 10 .409 14, .581 1, .00 11 .53 o
ATOM 507 N ASP A 35 21, .571 9, .765 12. .490 1. .00 11, .34 N
ATOM 509 CA ASP A 35 20 .224 9 .416 12. .794 1, .00 11 .07 C
ATOM 511 CB ASP A 35 19 .397 10 .677 12, .779 1 .00 12 .91 C
ATOM 514 CG ASP A 35 18 .231 10 .611 13, .697 1, .00 11 .93 C
ATOM 515 OD1 ASP A 35 17 .334 9 .791 13, .472 1, .00 12 .81 O
ATOM 516 OD2 ASP A 35 18 .166 11 .390 14, .700 1 .00 15 .59 O
ATOM 517 C ASP A 35 19 .687 8 .393 11, .816 1, .00 12 .34 C
ATOM 518 O ASP A 35 20 .470 7. .623 11, .250 1, .00 12 .33 o
ATOM 519 N THR A 36 18 .376 8, .385 11, .604 1, .00 12 .33 N
ATOM 521 CA THR A 36 17 .783 7, .395 10, .702 1, .00 13 .86 C
ATOM 523 CB THR A 36 16 .312 7 .198 10, .972 1, .00 13 .36 C
ATOM 525 OGl THR A 36 15 .603 8 .446 10, .753 1. .00 14 .42 O
ATOM 527 CG2 THR A 36 16 .058 6 .781 12, .383 1, .00 11 .98 C
ATOM 531 C THR A 36 17 .933 7 .710 9, .199 1, .00 14 .93 C
ATOM 532 O THR A 36 17 .341 7 .023 8 .379 1 .00 15 .02 o
ATOM 533 N GLY A 37 18 .699 8 .735 8, .885 1, .00 15 .27 N
ATOM 535 CA GLY A 37 18 .838 9 .282 7, .530 1, .00 15. .54 C
ATOM 538 C GLY A 37 18 .041 10 .594 7, .487 1, .00 16, .06 C
ATOM 539 O GLY A 37 17 .413 10 .973 8, .482 1, .00 14 .08 O
ATOM 540 N VAL A 38 18 .065 11 .292 6 .337 1 .00 16 .27 N
ATOM 542 CA VAL A 38 17 .315 12 .547 6, .177 1, .00 15 .20 C
ATOM 544 CB VAL A 38 18 .214 13 .784 6, .447 1, .00 15 .83 C
ATOM 546 CGI VAL A 38 17 .501 15 .075 6, .182 1, .00 16 .87 C ATOM 550 CG2 VAL A 38 18..774 13.,766 7.839 1..00 17.47 c
ATOM 554 C VAL A 38 16. ,863 12. ,628 4. 695 1. 00 16. 04 c
ATOM 555 O VAL A 38 17. ,622 12. ,277 3. 793 1. 00 14. 66 o
ATOM 556 N TYR A 39 15. 623 13. ,008 4. 532 1. 00 18. 21 N
ATOM 558 CA TYR A 39 15. ,046 13. .247 3. 214 1. ,00 19. ,90 C
ATOM 560 CB TYR A 39 13. ,564 13. ,150 3. 366 1. ,00 18. ,60 C
ATOM 563 CG TYR A 39 12. .795 13. ,480 2. 082 1. ,00 23. .59 C
ATOM 564 CDl TYR A 39 13. ,278 13. ,110 0. 833 1. .00 27. ,19 C
ATOM 566 CEl TYR A 39 12. .555 13. .413 -0. ,309 1. .00 30. .38 C
ATOM 568 CZ TYR A 39 11. .391 14. .129 -0. .226 1. ,00 31. .18 c
ATOM 569 OH TYR A 39 10. ,734 14. .412 -1. 434 1. ,00 30. ,34 o
ATOM 571 CE2 TYR A 39 10. ,912 14. ,528 1. 005 1. ,00 29. ,74 c
ATOM 573 CD2 TYR A 39 11. ,623 14. ,208 2. 144 1. ,00 23. ,70 c
ATOM 575 C TYR A 39 15. .495 14, .623 2. .786 1. .00 19. ,47 c
ATOM 576 O TYR A 39 14, .795 15 .631 2. .992 1. .00 22. .39 o
ATOM 577 N THR A 40 16, .675 14, .659 2. .240 1, .00 22. .23 N
ATOM 579 CA THR A 40 17, .366 15, .869 1. .904 1. .00 23. .84 C
ATOM 581 CB THR A 40 18, .797 15, .520 1. ,499 1. .00 25. .17 C
ATOM 583 OGl THR A 40 18, .841 14 .473 0, .518 1, .00 27, .20 O
ATOM 585 CG2 THR A 40 19, .633 14 .890 2, .687 1, .00 23, .66 c
ATOM 589 C THR A 40 16, .650 16 .659 0. .804 1, .00 25, .31 c
ATOM 590 O THR A 40 17, .008 17 .803 0. .566 1, .00 25, .93 o
ATOM 591 N SER A 41 15 .671 16 .051 0, .147 1 .00 25 .63 N
ATOM 593 CA SER A 41 14 .953 16 .703 -0. .942 1, .00 26, .05 C
ATOM 595 CB SER A 41 14 .662 15 .676 -2, ,047 1, .00 25, .60 C
ATOM 598 OG SER A 41 15. .836 15 .411 -2, .759 1, .00 26, .14 O
ATOM 600 C SER A 41 13, .669 17 .317 -0, .445 1, .00 26, .09 c
ATOM 601 O SER A 41 12 .896 17 .889 -1, .232 1 .00 26 .66 0
ATOM 602 N HIS A 42 13 .366 1 .179 0, .857 1. .00 22 .90 N
ATOM 604 CA HIS A 42 12 .245 17 .917 1, .419 1, .00 21, .28 C
ATOM 606 CB HIS A 42 12 .224 17 .792 2. .927 1, .00 21, .28 C
ATOM 609 CG HIS A 42 10 .988 18 .267 3, .562 1 .00 18 .79 C
ATOM 610 ND1 HIS A 42 10 .616 19 .591 3, .556 1 .00 17 .72 N
ATOM 612 CEl HIS A 42 9 .482 19 .706 4, .197 1 .00 14 .42 C
ATOM 614 NE2 HIS A 42 9 .124 18 .516 4, .654 1 .00 18, .07 N
ATOM 616 CD2 HIS A 42 10 .028 17 .601 4, .230 1, .00 15, .85 C
ATOM 618 C HIS A 42 12 .427 19 .379 1. .036 1 .00 20 .43 C
ATOM 619 O HIS A 42 13 .543 19 .890 1, .077 1 .00 19 .85 O
ATOM 620 N LEU A 43 11 .326 20 .044 0, .686 1 .00 21 .20 N
ATOM 622 CA LEU A 43 11 .380 21 .409 0, .210 1 .00 21, .25 C
ATOM 624 CB LEU A 43 10 .030 21 .945 -0, .087 1 .00 22, .55 C
ATOM 627 CG LEU A 43 9 .448 21 .512 -1 .433 1 .00 25 .81 C
ATOM 629 CDl LEU A 43 8 .021 21 .976 -1, .464 1 .00 27 .58 C
ATOM 633 CD2 LEU A 43 10 .234 22 .108 -2, .559 1 .00 27 .86 C
ATOM 637 C LEU A 43 12 .023 22 .311 1, .227 1 .00 21 .05 C
ATOM 638 O LEU A 43 12 .699 23 .255 0 .879 1 .00 18 .87 O
ATOM 639 N ASP A 44 11 .847 22 .042 2 .500 1 .00 21 .71 N
ATOM 641 CA ASP A 44 12 .514 22 .885 3 .487 1 .00 20 .87 C
ATOM 643 CB ASP A 44 11 .642 22 .917 4, .719 1. .00 21 .17 C
ATOM 646 CG ASP A 44 10 .262 23 .417 4, .441 1 .00 23 .00 C
ATOM 647 OD1 ASP A 44 10 .060 24 .154 3 .406 1 .00 21 .93 O
ATOM 648 OD2 ASP A 44 9 .325 23 .156 5 .206 1 .00 16 .28 O
ATOM 649 C ASP A 44 13 .962 22 .528 3 .812 1 .00 21 .02 C
ATOM 650 O ASP A 44 14 .593 23 .214 4, .604 1. .00 18 .05 O
ATOM 651 N LEU A 45 14 .488 21 .431 3 .252 1 .00 18 .30 N
ATOM 653 CA LEU A 45 15 .868 21 .070 3 .445 1 .00 19 .90 C
ATOM 655 CB LEU A 45 15 .922 19 .624 4 .024 1 .00 18 .80 C
ATOM 658 CG LEU A 45 15 .174 19 .394 5. .300 1 .00 18 .65 C
ATOM 660 CDl LEU A 45 15 .424 17 .925 5, .756 1 .00 18 .05 C
ATOM 664 CD2 LEU A 45 15 .714 20 .351 6 .357 1 .00 20 .33 c
ATOM 668 C LEU A 45 16 .750 21 .110 2 .197 1 .00 20 .37 c
ATOM 669 O LEU A 45 17 .960 20 .890 2 .251 1 .00 22 .13 0
ATOM 670 N ALA A 46 16 .104 21 .400 1 .079 1 .00 22 .51 N ATOM 672 CA ALA A 46 16.,728 21..327 -0.,210 1.00 21.,13 c
ATOM 674 CB ALA A 46 15. .738 21. .695 -1. 365 1. 00 21. ,15 c
ATOM 678 C ALA A 46 17. 880 22. .199 -0. 244 1. 00 20. ,28 c
ATOM 679 O ALA A 46 17. 806 23. .344 0. 177 1. 00 22. .66 o
ATOM 680 N GLY A 47 18. ,959 21. .639 -0. ,759 1. ,00 20. .67 N
ATOM 682 CA GLY A 47 20. ,217 22. .320 -0. ,968 1. ,00 21. .84 C
ATOM 685 C GLY A 47 21. ,042 22. .419 0. ,291 1. .00 21. ,53 C
ATOM 686 O GLY A 47 22. ,226 22. .881 0. ,280 1. .00 24. ,34 O
ATOM 687 N SER A 48 20. .522 21. .910 1. .392 1. .00 22, .52 N
ATOM 689 CA SER A 48 21. .310 21, .980 2. .610 1. ,00 21. .98 C
ATOM 691 CB . ASER A 48 20. ,384 22, .083 3. ,833 0. ,50 22. .54 C
ATOM 692 CB BSER A 48 20. ,408 22, .073 3. ,814 0. ,50 22. .26 C
ATOM 697 OG . ASER A 48 19. ,449 21. .001 3. ,944 0. ,50 23. ,88 O
ATOM 698 OG BSER A 48 19. .660 23, .258 3. .738 0. .50 21, ,10 O
ATOM 701 C SER A 48 22. .295 20, .852 2. .826 1. .00 21. ,47 C
ATOM 702 O SER A 48 23. .317 21, .066 3. .466 1. .00 21. .64 0
ATOM 703 N ALA A 49 22. ,035 19, .670 2. ,285 1. ,00 20. .96 N
ATOM 705 CA ALA A 49 22. ,951 18, .532 2. ,483 1. ,00 22. ,38 C
ATOM 707 CB ALA A 49 22, .192 17, .247 2. .068 1. .00 22, .64 C
ATOM 711 C ALA A 49 24, .233 18, .644 1. .718 1. .00 23, .21 C
ATOM 712 O ALA A 49 24. .228 18, .551 0. .500 1. .00 26. ,19 O
ATOM 713 N GLU A 50 25. .328 18, .866 2. .426 1. .00 20. ,40 N
ATOM 715 CA GLU A 50 26. .598 18 .826 1. .753 1. .00 21, ,76 C
ATOM 717 CB GLU A 50 27. .625 19 .892 2. .250 1. .00 22, ,32 C
ATOM 720 CG GLU A 50 27. .374 21 .244 1. .591 1. .00 26, ,62 C
ATOM 723 CD GLU A 50 28, .046 22 .421 2. .279 1. .00 30, ,59 C
ATOM 724 OE1 GLU A 50 28. .886 22, .227 3. .181 1. .00 24, ,43 O
ATOM 725 OE2 GLU A 50 27. .683 23 .561 1. .918 1. .00 35, ,88 O
ATOM 726 C GLU A 50 27. .208 17 .435 1, .866 1. .00 20, ,83 C
ATOM 727 O GLU A 50 28. .257 17 .188 1. .220 1. .00 21, ,81 O
ATOM 728 N GLN A 51 26, .761 16 .586 2. .783 1. .00 19, .08 N
ATOM 730 CA GLN A 51 27, .273 15 .186 2, .847 1. .00 18, .38 C
ATOM 732 CB GLN A 51 28, .416 14 .936 3. .863 1, .00 18 ,98 C
ATOM 735 CG GLN A 51 29, .720 15 .707 3, .698 1. .00 16, ,56 C
ATOM 738 CD GLN A 51 30, .864 15 .082 4, .418 1. .00 18, ,35 C
ATOM 739 OE1 GLN A 51 30, .728 14 .001 4. .993 1. .00 16, ,59 O
ATOM 740 NE2 GLN A 51 32 .021 15 .746 4, .421 1. .00 18 .88 N
ATOM 743 C GLN A 51 26 .048 14 .303 3, .122 1. .00 18 ,15 C
ATOM 744 O GLN A 51 25, .088 14 .739 3, .753 1. .00 17 ,95 O
ATOM 745 N CYS A 52 26, .032 13 .097 2. .549 1. .00 19 ,15 N
ATOM 747 CA CYS A 52 24, .933 12 .197 2. .657 1. .00 19, ,58 C
ATOM 749 CB CYS A 52 23 .994 12 .383 1, .463 1. .00 20 .58 C
ATOM 752 SG CYS A 52 22 .757 11 .113 1, .313 1, .00 23 .23 S
ATOM 753 C CYS A 52 25 .609 10 .827 2, .666 1, .00 19 ,62 C
ATOM 754 O CYS A 52 26 .112 10 .376 1, .630 1. .00 17 ,75 O
ATOM 755 N LYS A 53 25 .706 10 .188 3 .841 1 .00 18 .28 N
ATOM 757 CA LYS A 53 26 .435 8 .938 3 .934 1. .00 17 .30 C
ATOM 759 CB LYS A 53 27 .835 9 .165 4 .542 1, .00 17 .03 C
ATOM 762 CG LYS A 53 28 .733 10 .042 3, .720 1, .00 16 .05 C
ATOM 765 CD LYS A 53 30 .097 10 .281 4 .325 1, .00 17 .76 C
ATOM 768 CE LYS A 53 31 .031 11 .033 3 .333 1. .00 17 .09 C
ATOM 771 NZ LYS A 53 32 .138 11 .733 3 .893 1 .00 19 .33 N
ATOM 775 C LYS A 53 25 .698 7 .913 4 .801 1 .00 18 .20 C
ATOM 776 O LYS A 53 24 .966 8 .299 5 .712 1 .00 15 .09 O
ATOM 777 N ASP A 54 25 .905 6 .619 4 .518 1. .00 15 .90 N
ATOM 779 CA ASP A 54 25 .186 5 .563 5 .218 1 .00 17 .38 C
ATOM 781 CB ASP A 54 24 .244 4 .911 4 .223 1 .00 17 .91 C
ATOM 784 CG ASP A 54 23 .222 3 .960 4 .825 1 .00 20 .51 C
ATOM 785 OD1 ASP A 54 23 .261 3 .554 6 .029 1 .00 15 .70 O
ATOM 786 OD2 ASP A 54 22 .292 3 .563 4 .088 1 .00 19 .49 O
ATOM 787 C ASP A 54 26 .131 4 .552 5 .807 1 .00 17 .88 C
ATOM 788 O ASP A 54 26 .969 3 .968 5 .093 1 .00 17 .08 O
ATOM 789 N PHE A 55 25 .998 4 .356 7 .135 1 .00 15 .74 N ATOM 791 CA PHE A 55 26.,865 3..464 7.,867 1.,00 15..03 c
ATOM 793 CB PHE A 55 27. ,359 4. .168 9. ,131 1. ,00 13. ,99 c
ATOM 796 CG PHE A 55 28. 268 5. ,336 8. 844 1. 00 15. ,45 c
ATOM 797 CDl PHE A 55 27. 753 6. 544 8. 432 1. 00 15. ,76 c
ATOM 799 CEl PHE A 55 28. ,616 7. .657 8. ,155 1. ,00 14 , .10 c
ATOM 801 CZ PHE A 55 29. ,907 7, ,536 8. ,256 1. .00 13. .98 c
ATOM 803 CE2 PHE A 55 30. ,431 6. ,303 8. ,656 1. 00 15. .61 c
ATOM 805 CD2 PHE A 55 29. ,594 5. ,232 8. .950 1. 00 14. .62 c
ATOM 807 C PHE A 55 26. .160 2. .191 8. .265 1. ,00 15. .07 c
ATOM 808 O PHE A 55 26. .732 1. .387 9. ,025 1. .00 15. .44 o
ATOM 809 N THR A 56 24. .962 1. .994 7. ,769 1. ,00 15. .31 N
ATOM 811 CA THR A 56 24. ,149 0. .858 8. ,159 1. ,00 16. .77 c
ATOM 813 CB THR A 56 22. ,724 1. .253 8. ,463 1. ,00 17. .17 c
ATOM 815 OGl THR A 56 22. .006 1, .623 7. .272 1. .00 15, .48 0
ATOM 817 CG2 THR A 56 22. .628 2. .535 9. .443 1. .00 13 , .67 c
ATOM 821 C THR A 56 24. .134 -0. .328 7. .166 1. .00 20, ,28 c
ATOM 822 O THR A 56 23. .451 -1, .319 7. .407 1. ,00 21. .69 0
ATOM 823 N GLN A 57 24. .852 -0. .239 6. .069 1. .00 22 , .81 N
ATOM 825 CA GLN A 57 24, .736 -1. .314 5. .061 1. .00 25, .52 C
ATOM 827 CB GLN A 57 24, .681 -0, .724 3, .646 1. .00 25, .67 C
ATOM 830 CG GLN A 57 23, .521 0, .217 3. .502 1. .00 27, .65 c
ATOM 833 CD GLN A 57 23, .366 0, .800 2. .117 1. .00 36, .31 c
ATOM 834 OE1 GLN A 57 23 .871 0 .240 1, .156 1. .00 35 .97 o
ATOM 835 NE2 GLN A 57 22, .686 1 .938 2, .016 1. .00 30 .80 N
ATOM 838 C GLN A 57 25, .848 -2 .331 5. .196 1. .00 28 .75 C
ATOM 839 O GLN A 57 26. ,735 -2. ,182 6. .034 1. ,00 28, ,60 O
ATOM 840 N SER A 58 25. .792 -3. .388 4. .363 1. .00 32. ,27 N
ATOM 842 CA SER A 58 26, .798 -4. .440 4. .371 1. .00 34, .96 C
ATOM 844 CB SER A 58 26, .488 -5, .494 3. .291 1. .00 35, .23 C
ATOM 847 OG SER A 58 25, .088 -5, .548 3. .041 1. .00 37, .60 O
ATOM 849 C SER A 58 28, .149 -3, ,762 4. .140 1. .00 35, .56 C
ATOM 850 O SER A 58 29, .096 -3 .989 4, .843 1. .00 36, .58 O
ATOM 851 N ASN A 59 28, .224 -2 .889 3, .147 1, .00 37 .91 N
ATOM 853 CA ASN A 59 29, .409 -2 .054 3, .003 1. .00 38, .41 C
ATOM 855 CB ASN A 59 29, .288 -1, .232 1, .739 1. .00 39, .92 C
ATOM 858 CG ASN A 59 30, .172 -1, .727 0, .636 1, .00 44, .62 C
ATOM 859 OD1 ASN A 59 31 .413 -1 .752 0. .759 1, .00 52 .42 O
ATOM 860 ND2 ASN A 59 29 .547 -2 .121 -0. .468 1, .00 50 .97 N
ATOM 863 C ASN A 59 29, .421 -1 .061 4, .156 1, .00 37 .64 C
ATOM 864 O ASN A 59 28, .436 -0 .360 4, .338 1, .00 37 .26 O
ATOM 865 N PRO A 60 30, .474 -1 .028 4, .961 1, .00 37 .50 N
ATOM 866 CA PRO A 60 30 .591 -0 .066 6 .064 1 .00 36 .81 C
ATOM 868 CB PRO A 60 32 .016 -0 .315 6 .585 1 .00 37 .80 C
ATOM 871 CG PRO A 60 32 .661 -1. .116 5 .519 1 .00 38 .85 C
ATOM 874 CD PRO A 60 31 .589 -1 .986 4 .997 1 .00 38 .04 C
ATOM 877 C PRO A 60 30 .421 1 .431 5 .770 1 .00 35 .74 C
ATOM 878 O PRO A 60 30 .266 2 .188 6 .749 1 .00 34 .22 O
ATOM 879 N LEU A 61 30 .478 1 .876 4 .517 1 .00 33 .77 N
ATOM 881 CA LEU A 61 30 .183 3 .278 4 .258 1 .00 33 .86 C
ATOM 883 CB LEU A 61 31, .403 4 .170 4, .541 1, .00 34 .63 C
ATOM 886 CG LEU A 61 31 .122 5 .691 4 .652 1 .00 38 .34 C
ATOM 888 CDl LEU A 61 32 .418 6 .454 4 .765 1 .00 41 .14 C
ATOM 892 CD2 LEU A 61 30 .383 6 .236 3 .501 1 .00 40 .32 C
ATOM 896 C LEU A 61 29 .681 3 .440 2, .838 1, .00 31 .90 C
ATOM 897 O LEU A 61 30 .371 3 .109 1. .887 1. .00 32 .30 O
ATOM 898 N VAL A 62 28 .452 3 .862 2 .682 1 .00 29 .42 N
ATOM 900 CA VAL A 62 27 .944 4 .129 1 .363 1 .00 29 .40 C
ATOM 902 CB VAL A 62 26 .721 3 .365 1. .091 1 .00 28 .97 C
ATOM 904 CGI VAL A 62 26 .082 3 .877 -0 .187 1 .00 30 .81 C
ATOM 908 CG2 VAL A 62 27 .060 1 .874 1 .015 1 .00 31 .48 C
ATOM 912 C VAL A 62 27 .768 5 .625 1 .255 1 .00 27 .76 C
ATOM 913 O VAL A 62 27 .015 6 .233 1 .970 1 .00 26 .96 O
ATOM 914 N ASP A 63 28 .646 6 .224 0 .470 1 .00 28 .18 N ATOM 916 CA ASP A 63 28..600 7.643 0,.235 1..00 26.20 c
ATOM 918 CB ASP A 63 29, .993 8 .062 -0, .215 1, .00 26 .90 c
ATOM 921 CG ASP A 63 30. .222 9 .517 -0, .099 1, .00 25 .49 c
ATOM 922 OD1 ASP A 63 29. .290 10 .281 -0, .017 1. .00 26 .02 O
ATOM 923 OD2 ASP A 63 31, .318 10 .031 -0, .150 1. .00 28 .08 O
ATOM 924 C ASP A 63 27, .571 7 .929 -0, .826 1, .00 27 .79 c
ATOM 925 O ASP A 63 27, .455 7 .199 -1, .812 1, .00 27 .28 o
ATOM 926 N GLY A 64 26. .753 8 .936 -0, .581 1, .00 26 .48 N
ATOM 928 CA GLY A 64 25, .703 9 .316 -1. .502 1. .00 25 .76 C
ATOM 931 C GLY A 64 24, .357 8 .742 -1, .234 1. .00 25 .62 c
ATOM 932 O GLY A 64 23, .474 8 .881 -2, .053 1, .00 28 .14 0
ATOM 933 N SER A 65 24. .184 8 .096 -0, .080 1. .00 22 .83 N
ATOM 935 CA SER A 65 22. .953 7, .499 0, .304 1. .00 22. .85 C
ATOM 937 CB SER A 65 23, .003 6 .005 0, .117 1. .00 23 .23 C
ATOM 940 OG SER A 65 21, .699 5 .584 0, .027 1, .00 29 .80 O
ATOM 942 C SER A 65 22. .705 7 .773 1, .749 1, .00 21 .08 C
ATOM 943 O SER A 65 23, .671 7 .638 2, .504 1, .00 19 .41 O
ATOM 944 N CYS A 66 21. .521 8 .181 2, .140 1. .00 20 .00 N
ATOM 946 CA CYS A 66 21. .278 8 .539 3, .546 1, .00 20 .11 C
ATOM 948 CB CYS A 66 22, .034 9 .822 3, .885 1, .00 19 .63 C
ATOM 951 SG CYS A 66 21, .484 11 .254 2, .900 1, .00 19 .95 S
ATOM 952 C CYS A 66 19, .803 8 .601 3, .712 1, .00 18 .81 C
ATOM 953 O CYS A 66 19, .168 9 .468 4. .308 1, .00 17 .95 O
ATOM 954 N THR A 67 19. .180 7 .568 3, .214 1, .00 19 .39 N
ATOM 956 CA THR A 67 17. .768 7 .596 3, .075 1, .00 19 .72 C
ATOM 958 CB THR A 67 17. .481 6 .628 1, .924 1, .00 20 .77 C
ATOM 960 OGl THR A 67 18. .082 7, .189 0, .735 1, .00 26. .00 O
ATOM 962 CG2 THR A 67 16. .113 6 .443 1, .665 1, .00 23 .79 C
ATOM 966 C THR A 67 16. .941 7 .325 4, .315 1, .00 18 .14 C
ATOM 967 O THR A 67 17. .066 6 .297 4, .990 1, .00 16 .62 O
ATOM 968 N ASP A 68 16. .070 8, .278 4. .623 1. .00 17 .54 N
ATOM 970 CA ASP A 68 15, .191 8 .149 5, .786 1, .00 17 .68 C
ATOM 972 CB ASP A 68 14, .877 9 .530 6, .360 1, .00 16 .82 C
ATOM 975 CG ASP A 68 14. .131 9 .480 7. .697 1. .00 17 .49 C
ATOM 976 OD1 ASP A 68 13. .988 8, .380 8. .314 1. .00 14. .56 O
ATOM 977 OD2 ASP A 68 13. .610 10, .516 8. .221 1. .00 16. .31 O
ATOM 978 C ASP A 68 13. .909 7 .425 5. ,423 1, ,00 19 .14 C
ATOM 979 O ASP A 68 13. .100 7, .936 4. .626 1. .00 19 .83 O
ATOM 980 N ARG A 69 13. .688 6, .262 6. .023 1. .00 19, .38 N
ATOM 982 CA ARG A 69 12. .427 5, .549 5. .858 1. .00 20, .20 C
ATOM 984 CB ARG A 69 12. .665 4, .106 5. .357 1. .00 20, .57 C
ATOM 987 CG ARG A 69 13, .461 4 .061 4, .081 1, ,00 23 .84 C
ATOM 990 CD ARG A 69 13, .499 2 .688 3, .401 1, .00 28 .11 C
ATOM 993 NE ARG A 69 14, .384 2 .688 2, ,239 1, ,00 31 .97 N
ATOM 995 CZ ARG A 69 15, .683 2 .433 2. .284 1. .00 34 .34 C
ATOM 996 NHl ARG A 69 16 .288 2 .155 3 .437 1, .00 33 .57 N
ATOM 999 NH2 ARG A 69 16 .416 2 .464 1. .173 1, .00 37 .78 N
ATOM 1002 C ARG A 69 11, .615 5 .543 7, .120 1, .00 20 .20 C
ATOM 1003 O ARG A 69 10, .605 4 .861 7, .222 1, .00 19 .58 O
ATOM 1004 N GLN A 70 12, .022 6 .341 8. .120 1, .00 18 .56 N
ATOM 1006 CA GLN A 70 11 .359 6 .330 9. .404 1 .00 18 .67 C
ATOM 1008 CB GLN A 70 12 .459 6 .087 10, .480 1, .00 17 .38 C
ATOM 1011 CG GLN A 70 11. .887 5 .512 11, .734 1, .00 24 .45 C
ATOM 1014 CD GLN A 70 11, .094 6 .496 12, .618 1, .00 29 .69 C
ATOM 1015 OEl GLN A 70 11. .259 7 .719 12, .568 1, .00 28 .81 O
ATOM 1016 NE2 GLN A 70 10 .180 5 .934 13 .390 1 .00 36 .98 N
ATOM 1019 C GLN A 70 10 .678 7 .677 9, .729 1, .00 16 .99 C
ATOM 1020 O GLN A 70 9 .502 7. .745 10, .177 1, .00 18 .53 O
ATOM 1021 N GLY A 71 11, .448 8 .740 9, .546 1, .00 17 .00 N
ATOM 1023 CA GLY A 71 10 .936 10 .086 9 .792 1 .00 16 .91 C
ATOM 1026 C GLY A 71 11 .766 10 .862 10 .826 1 .00 16 .73 C
ATOM 1027 O GLY A 71 12 .023 12 .040 10 .683 1 .00 16 .08 O
ATOM 1028 N HIS A 72 12 .190 10 .148 11 .848 1 .00 15 .53 N ATOM 1030 CA HIS A 72 12,,902 10,.764 12.,965 1..00 14..82 c
ATOM 1032 CB HIS A 72 13, .305 9. .625 13. .926 1. ,00 14. .91 c
ATOM 1035 CG HIS A 72 13, .996 10. .088 15. ,170 1. ,00 11. .42 c
ATOM 1036 NDl HIS A 72 15, .356 10. .264 15. ,228 1. .00 11. .65 N
ATOM 1038 CEl HIS A 72 15, .690 10, .620 16. .456 1. .00 15. .57 C
ATOM 1040 NE2 HIS A 72 14, .603 10, .660 17. .194 1. .00 12. .24 N
ATOM 1042 CD2 HIS A 72 13, .527 10, .309 16. ,414 1. .00 15. .18 C
ATOM 1044 C HIS A 72 14, ,077 11. .632 12. ,515 1. .00 14. .31 C
ATOM 1045 O HIS A 72 14, .157 12. .811 12, ,906 1. .00 14. .46 O
ATOM 1046 N GLY A 73 14, .993 11, .101 11, .686 1. .00 13, .23 N
ATOM 1048 CA GLY A 73 16, .140 11, .851 11. .227 1. .00 13, .67 C
ATOM 1051 C GLY A 73 15, .743 13, .097 10. .452 1. .00 14. .47 C
ATOM 1052 O GLY A 73 16, .388 14. .147 10. .556 1. .00 14. .58 O
ATOM 1053 N THR A 74 14. .691 12, .976 9. .638 1. ,00 14, .43 N
ATOM 1055 CA THR A 74 14 .223 14 .163 8. .902 1. .00 14. .75 C
ATOM 1057 CB THR A 74 13 .166 13 .722 7, .889 1, .00 15 .14 C
ATOM 1059 OGl THR A 74 13 .832 12 .851 6, .979 1. .00 14, .14 O
ATOM 1061 CG2 THR A 74 12 .703 14, .949 7. ,052 1. .00 17, .07 C
ATOM 1065 C THR A 74 13 .672 15 .256 9. .779 1. .00 13 .89 C
ATOM 1066 O THR A 74 13 .964 16 .449 9, .549 1, .00 14. .36 O
ATOM 1067 N HIS A 75 12 .985 14 .834 10. .823 1, .00 13, .97 N
ATOM 1069 CA HIS A 75 12 .345 15 .653 11, ,803 1. .00 13, .53 C
ATOM 1071 CB HIS A 75 11 .464 1 .793 12, .693 1 .00 14 .00 C
ATOM 1074 CG HIS A 75 10 .525 15 .543 13, .566 1, .00 13 .88 C
ATOM 1075 NDl HIS A 75 10 .923 16 .209 14, .706 1, .00 13, .19 N
ATOM 1077 CEl HIS A 75 9 .888 16 .830 15, .235 1, .00 15, .22 C
ATOM 1079 NE2 HIS A 75 8 .826 16, .616 14, .465 1, .00 15, .33 N
ATOM 1081 CD2 HIS A 75 9 .203 15 .822 13, ,415 1, .00 14 .61 C
ATOM 1083 C HIS A 75 13 .464 16 .423 12, .565 1, .00 14, .20 C
ATOM 1084 O HIS A 75 13 .447 17 .650 12, .685 1, .00 11, .71 O
ATOM 1085 N VAL A 76 14 .436 15 .685 13, .031 1, .00 13, .61 N
ATOM 1087 CA VAL A 76 15 .543 16 .273 13, .761 1, .00 13 .90 C
ATOM 1089 CB VAL A 76 16 .471 15 .117 14, .276 1 .00 12 .59 C
ATOM 1091 CGI VAL A 76 17 .771 15 .657 14, .716 1, .00 13 .85 c
ATOM 1095 CG2 VAL A 76 15 .788 14, .354 15, .381 1, .00 13, .36 c
ATOM 1099 C VAL A 76 16 .280 17, .319 12, .925 1, .00 13, .76 c
ATOM 1100 O VAL A 76 16 .549 18 .419 13, .362 1, .00 13 .81 o
ATOM 1101 N ALA A 77 16 .598 16 .976 11, .693 1, .00 13, .31 N
ATOM 1103 CA ALA A 77 17 .316 17 .850 10, .844 1, .00 13, .43 C
ATOM 1105 CB ALA A 77 17, .586 17, .164 9, ,553 1, .00 12, .82 C
ATOM 1109 C ALA A 77 16, .538 19, .154 10, ,631 1, .00 13, .42 C
ATOM 1110 O ALA A 77 17 .137 20 .256 10, .595 1, .00 16 .51 O
ATOM 1111 N GLY A 78 15 .223 19 .047 10, .501 1, .00 13 .22 N
ATOM 1113 CA GLY A 78 14 .413 20 .237 10, .270 1, .00 14, .59 C
ATOM 1116 C GLY A 78 14 .431 21, .221 11, ,448 1, .00 14, .40 C
ATOM 1117 O GLY A 78 1 .427 22 .440 11, .294 1 .00 14 .85 O
ATOM 1118 N THR A 79 14 .537 20 .673 12, .643 1, .00 12 .64 N
ATOM 1120 CA THR A 79 14 .546 21 .535 13, .817 1, .00 11, .83 C
ATOM 1122 CB THR A 79 14 .350 20 .656 15, .063 1, .00 11, .71 C
ATOM 1124 OGl THR A 79 12 .990 20, .162 15, .166 1, .00 12, .60 O
ATOM 1126 CG2 THR A 79 14 .569 21 .491 16, .347 1 .00 11 .10 C
ATOM 1130 C THR A 79 15 .842 22 .248 13, .795 1. .00 12, .20 C
ATOM 1131 O THR A 79 15 .917 23 .440 14, .122 1, .00 12, .14 O
ATOM 1132 N VAL A 80 16 .917 21 .568 13, .358 1, .00 11, .44 N
ATOM 1134 CA VAL A 80 18 .195 22 .225 13, .293 1, .00 11 .80 C
ATOM 1136 CB VAL A 80 19 .299 21 .273 12 .865 1 .00 11 .91 C
ATOM 1138 CGI VAL A 80 20 .637 21 .963 12, .687 1, .00 13, .34 C
ATOM 1142 CG2 VAL A 80 19 .520 20 .158 13, .884 1, .00 12, .77 C
ATOM 1146 C VAL A 80 18 .216 23 .369 12, .266 1, .00 13, .39 C
ATOM 1147 O VAL A 80 18 .646 24 .514 12 .553 1 .00 12 .65 o
ATOM 1148 N LEU A 81 17 .751 23 .054 11, .069 1 .00 13 .69 N
ATOM 1150 CA LEU A 81 18 .057 23 .946 9 .965 1 .00 14 .13 C
ATOM 1152 CB LEU A 81 19 .454 23 .675 9 .439 1 .00 14 .13 C ATOM 1155 CG LEU A 81 19,.893 22.189 9.225 1 . 00 11 . 54 C
ATOM 1157 CDl LEU A 81 19, .058 21 .552 8.105 1 . 00 15 . 65 C
ATOM 1161 CD2 LEU A 81 21, ,308 22 .059 8.854 1 . 00 14 . 83 C
ATOM 1165 C LEU A 81 17. ,043 24 .065 8.827 1 . 00 15 . 14 C
ATOM 1166 O LEU A 81 17, .442 24 .518 7.766 1 . 00 17 . 76 O
ATOM 1167 N ALA A 82 15, ,791 23 .694 9.035 1 00 15.38 N
ATOM 1169 CA ALA A 82 14, .830 23 .894 7.920 1 00 16.54 C
ATOM 1171 CB ALA A 82 13. .485 23 .412 8.253 1 00 16.56 C
ATOM 1175 C ALA A 82 14, .807 25 .381 7.616 1 00 17.73 C
ATOM 1176 O ALA A 82 14, .873 26 .246 8.522 1 00 16.11 O
ATOM 1177 N HIS A 83 14, ,637 25 .678 6.321 1.00 18.61 N
ATOM 1179 CA HIS A 83 14, ,802 27 .048 5.845 1 .00 17 .82 C
ATOM 1181 CB HIS A 83 16, .057 27 .116 4.996 1 .00 18 ,66 C
ATOM 1184 CG HIS A 83 16, ,040 26 .187 3.831 1 .00 19 .55 C
ATOM 1185 NDl HIS A 83 14 .935 26 .066 023 1 .00 23 .39 N
ATOM 1187 CEl HIS A 83 15 .196 25 .205 056 1 .00 24 .12 C
ATOM 1189 NE2 HIS A 83 16 .395 24 .706 259 1 .00 24 .21 N
ATOM 1191 CD2 HIS A 83 16, .960 25 .326 349 1 .00 22 .73 C
ATOM 1193 C HIS A 83 13 .606 27 .689 119 1 .00 19 .24 C
ATOM 1194 O HIS A 83 13 .802 28 .694 468 1 .00 20 .21 O
ATOM 1195 N GLY A 84 12 .433 27 .158 342 1 .00 19 .88 N
ATOM 1197 CA GLY A 84 11, .151 27 .653 874 1 .00 23 .01 C
ATOM 1200 C GLY A 84 10 .891 27 .373 388 1 .00 23 .83 C
ATOM 1201 O GLY A 84 9 .816 27 .693 873 1 .00 25 .88 O
ATOM 1202 N GLY A 85 11. .891 26 .852 716 1 .00 25 .14 N
ATOM 1204 CA GLY A 85 11. .754 26 .452 333 1 .00 28 .31 C
ATOM 1207 C GLY A 85 11, ,845 27 .607 0.361 1 .00 30 .82 C
ATOM. 1208 O GLY A 85 11, .704 28 .777 0.750 1 .00 32 .32 O
ATOM 1209 N SER A 86 12, .066 27 .253 0.910 1 .00 33 .23 N
ATOM 1211 CA SER A 86 12, .332 28 .220 1.982 1 .00 35 .37 C
ATOM 1213 CB SER A 86 12, .287 27 .551 3.374 1 .00 35 .51 C
ATOM 1216 OG SER A 86 11, ,022 26 .920 3.531 1 .00 36 .16 O
ATOM 1218 C SER A 86 11, .323 29 .323 984 1 .00 35 .85 C
ATOM 1219 O SER A 86 11, ,673 30 .481 155 1 .00 37 .71 O
ATOM 1220 N ASN A 87 10. .066 28 .993 784 1 .00 36. .18 N
ATOM 1222 CA ASN A 87 9. .060 30, .024 844 1 .00 37, .05 C
ATOM 1224 CB ASN A 87 7, .842 29 .472 589 1 .00 37 .33 C
ATOM 1227 CG ASN A 87 6, ,943 28 .626 1.702 1 .00 40 .53 C
ATOM 1228 OD1 ASN A 87 7. ,323 28 .240 0.581 1 .00 40, .83 O
ATOM 1229 ND2 ASN A 87 5. .732 28 .329 2.205 1 .00 39, .22 N
ATOM 1232 C ASN A 87 8. .678 30 .600 0.469 1 .00 35, .97 C
ATOM 1233 O ASN A 87 7, .564 31 .143 0.295 1 .00 36 .81 O
ATOM 1234 N GLY A 88 9, .554 30 .402 0.526 1 .00 34 .13 N
ATOM 1236 CA GLY A 88 9, .307 30 .979 841 1 .00 32 .36 C
ATOM 1239 C GLY A 88 8. ,149 30 .556 701 1 .00 30 .27 C
ATOM 1240 O GLY A 88 7, .882 31 .189 728 1 .00 30 .72 O
ATOM 1241 N GLN A 89 7. .375 29 .545 305 1 .00 28 .00 N
ATOM 1243 CA GLN A 89 6, .212 29 .190 117 1 .00 27 .11 C
ATOM 1245 CB GLN A 89 4. .898 29 .091 269 1 .00 28 .51 C
ATOM 1248 CG GLN A 89 3, .596 28 .969 114 0 .10 26 .48 C
ATOM 1251 CD GLN A 89 2 .269 28 .881 318 0 .10 25 .84 C
ATOM 1252 OEl GLN A 89 2, .243 28 .873 085 0 .10 20 .77 O
ATOM 1253 NE2 GLN A 89 ,1. .164 28 .811 052 0 .10 23 .86 N
ATOM 1256 C GLN A 89 6, .384 27 .908 974 1 .00 26 .64 C
ATOM 1257 O GLN A 89 5 .463 27 .490 638 1 .00 25 .73 O
ATOM 1258 N GLY A 90 7. .572 27 .312 967 1 .00 26 .19 N
ATOM 1260 CA GLY A 90 7, .781 26 .104 760 1.00 24.89 C
ATOM 1263 C GLY A 90 8, .133 26 .372 6.223 1.00 25.02 C
ATOM 1264 O GLY A 90 7, .940 27 .492 6.751 00 25.06 O
ATOM 1265 N VAL A 91 8 .598 25 .330 6.888 00 22.83 N
ATOM 1267 CA VAL A 91 8 .942 25 .462 8.304 00 20.77 C
ATOM 1269 CB VAL A 91 8 .681 24 .116 9.045 00 19.91 C
ATOM 1271 CGI VAL A 91 9 .781 23 .160 8.797 00 21.25 C ATOM 1275 CG2 VAL A 91 8..463 24,.309 10.,528 1..00 21,,36 c
ATOM 1279 C VAL A 91 10. .344 25, .938 8. ,411 1. ,00 18, ,60 c
ATOM 1280 O VAL A 91 11. .184 25, .738 7. ,532 1. .00 19, ,96 o
ATOM 1281 N TYR A 92 10. ,632 26. .589 9. ,547 1. ,00 18, ,11 N
ATOM 1283 CA TYR A 92 11. .941 27, .076 9. .868 1. .00 17 .39 C
ATOM 1285 CB TYR A 92 11. .880 28, .546 10. .256 1. .00 16, ,87 C
ATOM 1288 CG TYR A 92 11. .827 29, .420 9. .027 1. .00 17, ,86 C
ATOM 1289 CDl TYR A 92 12. .989 29, .758 8. .379 1. .00 16, ,53 C
ATOM 1291 CEl TYR A 92 12. .993 30, .516 7. .233 1. .00 20 .55 C
ATOM 1293 CZ TYR A 92 11. .793 30, .963 6. .717 1. .00 23 ,54 c
ATOM 1294 OH TYR A 92 11. .862 31, .737 5. ,549 1. .00 25, ,77 o
ATOM 1296 CE2 TYR A 92 10. .619 30, .672 7. ,325 1. .00 19, ,89 c
ATOM 1298 CD2 TYR A 92 10. .626 29. .840 8. ,507 1. .00 20, ,04 c
ATOM 1300 C TYR A 92 12, .542 26, .375 11. .099 1. .00 14 .56 c
ATOM 1301 O TYR A 92 11, .856 26 .154 12, .042 1, .00 14 .48 o
ATOM 1302 N GLY A 93 13, .824 26 .133 11. .058 1. .00 13 .49 N
ATOM 1304 CA GLY A 93 14, .547 25, .546 12. .182 1. .00 15 .74 C
ATOM 1307 C GLY A 93 15, .350 26, .635 12. .819 1. .00 14 .86 C
ATOM 1308 O GLY A 93 15, .203 27 .819 12. .473 1. .00 17 .02 O
ATOM 1309 N VAL A 94 16, .231 26 .278 13. .759 1, .00 14 .32 N
ATOM 1311 CA VAL A 94 16, .981 27 .306 14. .421 1, .00 1 .34 C
ATOM 1313 CB VAL A 94 17, .654 26, .753 15. .712 1, .00 13 .59 C
ATOM 1315 CGI VAL A 94 18 .263 27 .876 16, .515 1 .00 13 .33 C
ATOM 1319 CG2 VAL A 94 16, .633 26 .043 16, .538 1, .00 13 .90 C
ATOM 1323 C VAL A 94 18, .010 28 .055 13, .577 1, .00 14 .53 C
ATOM 1324 O VAL A 94 18, .196 29 .258 13. .779 1, .00 16 .71 O
ATOM 1325 N ALA A 95 18. .724 27, .356 12. .692 1, .00 14 .24 N
ATOM 1327 CA ALA A 95 19, .859 27 .839 11, .990 1, .00 14 .00 C
ATOM 1329 CB ALA A 95 21, .100 27 .227 12, .574 1, .00 14 .43 C
ATOM 1333 C ALA A 95 19, .757 27 .491 10. .498 1, .00 15 .52 c
ATOM 1334 O ALA A 95 20, .476 26, .644 9. .954 1, .00 13 .66 o
ATOM 1335 N PRO A 96 18, .847 28 .184 9. .840 1, .00 15 .59 N
ATOM 1336 CA PRO A 96 18, .487 27 .876 8. .443 1, .00 16 .93 C
ATOM 1338 CB PRO A 96 17, .330 28 .851 8. .170 1, .00 17 .23 C
ATOM 1341 CG PRO A 96 17. .628 29, .986 9. .086 1. .00 16 .22 C
ATOM 1344 CD PRO A 96 18. .078 29, .307 10. .383 1. .00 13, .71 C
ATOM 1347 C PRO A 96 19. .598 28 .049 7. .403 1. .00 17 .82 C
ATOM 1348 O PRO A 96 19, .478 27, .477 6. .306 1. .00 16 .77 O
ATOM 1349 N GLN A 97 20. .664 28, .772 7. .719 1. .00 17 .73 N
ATOM 1351 CA GLN A 97 21. .812 28, .891 6. .826 1. .00 18 .02 C
ATOM 1353 CB GLN A 97 22. .374 30, .341 6. .726 1. .00 18, .03 C
ATOM 1356 CG GLN A 97 21, .509 31 .218 5. ,783 1. .00 22 .97 c
ATOM 1359 CD GLN A 97 20, .220 31 .715 6, .401 1. .00 23 .17 c
ATOM 1360 OEl GLN A 97 20, .303 32 .467 7. .345 1. .00 27 .41 o
ATOM 1361 NE2 GLN A 97 19, .016 31 .311 5. .865 1, .00 26 .08 N
ATOM 1364 C GLN A 97 22 .901 27 .903 7, .080 1, .00 17 .35 C
ATOM 1365 O GLN A 97 23 .900 27 .913 6, .351 1, .00 17 .54 O
ATOM 1366 N ALA A 98 22 .763 27 .057 8. .125 1, .00 15 .56 N
ATOM 1368 CA ALA A 98 23, .794 26 .040 8, .361 1, .00 16 .07 C
ATOM 1370 CB ALA A 98 23, .615 25 .387 9, .738 1, .00 15 .36 C
ATOM 1374 C ALA A 98 23 .657 24 .997 7. .256 1 .00 15 .80 C
ATOM 1375 O ALA A 98 22 .610 24 .906 6. .610 1. .00 18 .34 O
ATOM 1376 N LYS A 99 24 .683 24 .195 7. .082 1, .00 16 .19 N
ATOM 1378 CA LYS A 99 24, .670 23 .108 6, .118 1, .00 15 .45 C
ATOM 1380 CB LYS A 99 25, .882 23 .152 5, .268 1 .00 16 .64 C
ATOM 1383 CG LYS A 99 25 .789 24 .264 4, .222 1 .00 17 .86 C
ATOM 1386 CD LYS A 99 24 .616 24 .101 3, .322 1 .00 22 .94 C
ATOM 1389 CE LYS A 99 24, .844 25 .062 2. .185 1, .00 28 .36 C
ATOM 1392 NZ LYS A 99 23, .614 25 .181 1. .383 1, .00 28 .94 N
ATOM 1396 C LYS A 99 24 .604 21 .759 6 .887 1 .00 15 .54 C
ATOM 1397 O LYS A 99 25 .012 21 .684 8. .019 1 .00 15 .10 O
ATOM 1398 N LEU A 100 24 .136 20 .742 6 .185 1 .00 14 .73 N
ATOM 1400 CA LEU A 100 23 .801 19 .460 6 .791 1 .00 15 .28 C ATOM 1402 CB LEU A 100 22,.361 19,.131 6..489 1..00 14,,80 c
ATOM 1405 CG LEU A 100 21, .852 17, .724 6. .719 1. .00 16, .65 c
ATOM 1407 CDl LEU A 100 21, .751 17, .484 8. .242 1. .00 17, ,43 c
ATOM 1411 CD2 LEU A 100 20, .500 17, .473 6. .155 1. .00 17, ,01 c
ATOM 1415 C LEU A 100 24, .743 18 .373 6, .336 1, .00 15 .64 c
ATOM 1416 O LEU A 100 25, .114 18, .290 5, .154 1. .00 17 ,60 o
ATOM 1417 N TRP A 101 25, .206 17, .566 7, .298 1. .00 15 ,09 N
ATOM 1419 CA TRP A 101 25, .895 16, .350 6. .942 1. .00 15, ,10 C
ATOM 1421 CB TRP A 101 27, .265 16, .234 7, .534 1. .00 14. .66 C
ATOM 1424 CG TRP A 101 28. .408 17 .171 7, .076 1, .00 13 .68 c
ATOM 1425 CDl TRP A 101 28, .342 18 .164 6, .137 1. .00 14 .24 c
ATOM 1427 NEl TRP A 101 29, .575 18, .741 5, .956 1. .00 14 .73 N
ATOM 1429 CE2 TRP A 101 30, .465 18, .110 6, .770 1. .00 14 .20 C
ATOM 1430 CD2 TRP A 101 29 .751 17 .123 7 .498 1, .00 15 .01 C
ATOM 1431 CE3 TRP A 101 30, .470 16, .329 8. .413 1. .00 15. .44 C
ATOM 1433 CZ3 TRP A 101 31, .791 16, .598 8. .605 1. .00 14, .31 C
ATOM 1435 CH2 TRP A 101 32. .451 17, .587 7. .845 1. .00 15, .17 C
ATOM 1437 CZ2 TRP A 101 31. .780 18, .363 6. .977 1. ,00 14, .09 C
ATOM 1439 C TRP A 101 24, .932 15, .267 7. .451 1. .00 16 .46 C
ATOM 1440 O TRP A 101 24, .830 15, .022 8, .675 1. .00 14 .81 o
ATOM 1441 N ALA A 102 24, .250 14, .579 6. .534 1. .00 14. .78 N
ATOM 1443 CA ALA A 102 23. .255 13. .610 6. .910 1. .00 15, .54 C
ATOM 1445 CB ALA A 102 22, .086 13, .639 5, .973 1, .00 16 .18 C
ATOM 1449 C ALA A 102 23, .897 12, .221 6. .941 1. .00 15 .83 C
ATOM 1450 O ALA A 102 24, .187 11, .661 5, .898 1. .00 15. .17 O
ATOM 1451 N TYR A 103 24, .148 11, .692 8. .140 1. .00 14, .35 N
ATOM 1453 CA TYR A 103 24, .797 10, .400 8. .290 1. ,00 14, .35 C
ATOM 1455 CB TYR A 103 25, .985 10, .493 9. .225 1. .00 13 .92 C
ATOM 1458 CG TYR A 103 27, .247 11, .147 8. .697 1. .00 13 .29 C
ATOM 1459 CDl TYR A 103 27, .275 11, .938 7, .550 1. .00 14 .70 C
ATOM 1461 CEl TYR A 103 28, .455 12, .512 7. .113 1. .00 14 .99 C
ATOM 1463 CZ TYR A 103 29, .587 12, .335 7. .783 1. .00 13 .42 C
ATOM 146.4 OH TYR A 103 30. .820 12 .886 7, .417 1. .00 17 .55 O
ATOM 1466 CE2 TYR A 103 29, .608 11, .561 8, .961 1. .00 11 .55 c
ATOM 1468 CD2 TYR A 103 28, .445 10, .996 9, .399 1. .00 12 .45 c
ATOM 1470 C TYR A 103 23, .813 9, .419 8, .860 1. .00 13 .70 c
ATOM 1471 O TYR A 103 23, .336 9 .583 9, .966 1, .00 13 .01 0
ATOM 1472 N LYS A 104 23, .490 8 .383 8, .101 1. .00 13 .67 N
ATOM 1474 CA LYS A 104 22, .524 7 .385 8, .564 1. .00 12 .46 C
ATOM 1476 CB LYS A 104 21, .773 6, .738 7, .407 1. .00 14 .90 C
ATOM 1479 CG LYS A 104 20, .789 5. .718 7, .815 1, .00 15 .07 c
ATOM 1482 CD LYS A 104 19 .991 5 .144 6 .616 1. .00 14 .19 c
ATOM 1485 CE LYS A 104 18 .751 4 .402 7. .036 1, .00 17 .35 c
ATOM 1488 NZ LYS A 104 18 .027 3 .784 5 .831 1, .00 15 .18 N
ATOM 1492 C LYS A 104 23 .249 6 .327 9, .362 1, .00 14 .29 C
ATOM 1493 O LYS A 104 2 .138 5 .652 8 .836 1 .00 14 .08 O
ATOM 1494 N VAL A 105 2 .893 6 .215 10 .645 1 .00 12 .75 N
ATOM 1496 CA VAL A 105 23 .513 5 .287 11 .592 1 .00 13 .70 C
ATOM 1498 CB VAL A 105 24 .301 6 .043 12 .684 1, .00 13 .12 C
ATOM 1500 CGI VAL A 105 25 .244 6 .961 12 .010 1, .00 14 .29 C
ATOM 1504 CG2 VAL A 105 23 .388 6 .804 13 .578 1. .00 12 .47 C
ATOM 1508 C VAL A 105 22 .491 4 .405 12 .292 1 .00 13 .35 C
ATOM 1509 O VAL A 105 22 .851 3 .501 13 .036 1 .00 14 .40 O
ATOM 1510 N LEU A 106 21 .218 4 .733 12 .140 1 .00 13 .80 N
ATOM 1512 CA LEU A 106 20 .133 3 .912 12 .678 1. .00 15 .31 C
ATOM 1514 CB LEU A 106 19 .165 4 .715 13 .533 1 .00 14 .78 C
ATOM 1517 CG LEU A 106 19 .820 5 .395 14 .752 1. .00 14 .84 C
ATOM 1519 CDl LEU A 106 18 .745 6 .216 15 .434 1 .00 13 .44 C
ATOM 1523 CD2 LEU A 106 20 .365 4 .281 15 .645 1 .00 13 .80 C
ATOM 1527 C LEU A 106 19 .328 3 .395 11 .488 1 .00 16 .71 C
ATOM 1528 O LEU A 106 19 .217 4 .083 10 .457 1 .00 14 .73 O
ATOM 1529 N GLY A 107 18 .812 2 .184 11 .617 1 .00 18 .94 N
ATOM 1531 CA GLY A 107 17 .950 1 .629 10 .581 1, .00 20 .89 C ATOM 1534 C GLY A 107 16.,534 2,.176 10..597 1..00 22..18 c
ATOM 1535 O GLY A 107 16. ,136 3. .087 11. .335 1. .00 21. .65 o
ATOM 1536 N ASP A 108 15. ,714 1. .570 9. ,755 1. .00 24, .79 N
ATOM 1538 CA ASP A 108 14. ,419 2, .139 9. ,442 1. .00 25. ,79 C
ATOM 1540 CB ASP A 108 13. .946 1, ,584 8. .117 1, .00 26, .09 C
ATOM 1543 CG ASP A 108 14. .971 1, .774 7. .022 1. .00 28, .51 C
ATOM 1544 OD1 ASP A 108 15. ,795 2. .721 7. .082 1, ,00 27, .80 O
ATOM 1545 OD2 ASP A 108 15. ,020 1. .038 6. .025 1. .00 30, .77 O
ATOM 1546 C ASP A 108 13, .331 1, .997 10. .489 1. .00 26, .41 C
ATOM 1547 O ASP A 108 12. .229 2 .535 10, ,294 1, .00 26 .78 O
ATOM 1548 N ASN A 109 13. .629 1, .262 11. ,566 1. .00 26 .19 N
ATOM 1550 CA ASN A 109 12. .751 1, .172 12. ,719 1. .00 25, .53 C
ATOM 1552 CB ASN A 109 12. .399 -0, .264 13. .022 1, ,00 26, .84 C
ATOM 1555 CG ASN A 109 11, .599 -0 .920 11, .863 1 .00 30 .82 C
ATOM 1556 OD1 ASN A 109 10. .606 -0, .338 11. .353 1. ,00 35, .24 O
ATOM 1557 ND2 ASN A 109 12. ,038 -2, .093 11. .429 1. .00 36, .51 N
ATOM 1560 C ASN A 109 13. ,340 1, .943 13. .933 1. .00 24, .23 C
ATOM 1561 O ASN A 109 12, ,941 1, .773 15. .071 1. .00 23. .68 O
ATOM 1562 N GLY A 110 14. .325 2, .774 13. .652 1, ,00 22 .01 N
ATOM 1564 CA GLY A 110 14. .823 3, .684 14. .685 1, .00 20 .58 C
ATOM 1567 C GLY A 110 15. .783 3, .001 15. .656 1, .00 18, .79 C
ATOM 1568 O GLY A 110 15. .989 3, .492 16. .797 1. .00 18, .63 O
ATOM 1569 N SER A 111 16. .373 1 .885 15, .208 1, .00 16 .52 N
ATOM 1571 CA SER A 111 17. .377 1 .225 16, .057 1, .00 17 .48 C
ATOM 1573 CB SER A 111 16. .805 0. .002 16. .752 1. .00 18 .93 C
ATOM 1576 OG SER A 111 16. .663 -1, .046 15. .856 1. .00 20, .57 O
ATOM 1578 C SER A 111 18. ,625 0, .916 15. .250 1, .00 15, .84 C
ATOM 1579 O SER A 111 18. ,585 0 .814 14, .022 1. .00 15 .08 O
ATOM 1580 N GLY A 112 19, ,767 0 .761 15, .913 1, .00 15 .46 N
ATOM 1582 CA GLY A 112 20. .991 0 .537 15, .198 1, .00 14 .74 C
ATOM 1585 C GLY A 112 22. .080 -0, .033 16. .063 1. .00 13 .99 C
ATOM 1586 O GLY A 112 21, .852 -0 .365 17, .219 1, .00 12. .44 O
ATOM 1587 N TYR A 113 23, .229 -0 .171 15, .431 1, .00 13 .65 N
ATOM 1589 CA TYR A 113 24, .372 -0 .836 16, .000 1, .00 14 .69 C
ATOM 1591 CB TYR A 113 24, .992 -1. .754 14, ,982 1, .00 14 .83 C
ATOM 1594 CG TYR A 113 24. .139 -2, .928 14, .627 1, .00 16. .70 C
ATOM 1595 CDl TYR A 113 24, .217 -4 .098 15, .339 1. .00 18 .38 C
ATOM 1597 CEl TYR A 113 23, .392 -5 .213 14, .997 1, .00 18 .28 C
ATOM 1599 CZ TYR A 113 22, .516 -5 .121 13, .926 1, .00 19 .64 C
ATOM 1600 OH TYR A 113 21, .730 -6 .214 13, .573 1, .00 20 .11 O
ATOM 1602 CE2 TYR A 113 22, .465 -3 .958 13, .189 1, .00 19 .66 c
ATOM 1604 CD2 TYR A 113 23 .269 -2 .871 13 .557 1. .00 15 .59 c
ATOM 1606 C TYR A 113 25 .441 0 .094 16 .471 1 .00 13 .86 c
ATOM 1607 O TYR A 113 25 .825 1 .019 15 .758 1 .00 14 .60 o
ATOM 1608 N SER A 114 25, .880 -0 .113 17, .706 1 .00 13 .30 N
ATOM 1610 CA SER A 114 26 .958 0 .655 18 .290 1 .00 13 .35 c
ATOM 1612 CB SER A 114 27 .507 -0 .213 19 .432 1 .00 15 .78 c
ATOM 1615 OG SER A 114 28 .722 0 .270 19 .995 1 .00 14 .15 o
ATOM 1617 C SER A 114 28 .125 0 .916 17 .339 1 .00 14 .05 c
ATOM 1618 O SER A 114 28 .615 2 .023 17 .226 1. .00 13 .89 0
ATOM 1619 N ASP A 115 28 .581 -0 .139 16 .665 1 .00 13 .29 N
ATOM 1621 CA ASP A 115 29 .724 0 .014 15 .754 1 .00 14 .72 C
ATOM 1623 CB AASP A 115 30 .159 -1 .299 15 .125 0 .50 16 .85 C
ATOM 1624 CB BASP A 115 29 .949 -1 .288 14 .969 0 .50 15 .71 C
ATOM 1629 CG AASP A 115 29 .100 -1 .900 14 .308 0 .50 20 .25 C
ATOM 1630 CG BASP A 115 30 .701 -2 .324 15 .752 0 .50 16 .91 C
ATOM 1631 OD1AASP A 115 28 .555 -1 .177 13 .460 0 .50 32 .11 o
ATOM 1632 OD1BASP A 115 31 .693 -1 .951 16 .379 0 .50 24 .07 o
ATOM 1633 OD2AASP A 115 28 .717 -3 .057 14 .477 0 .50 33 .25 o
ATOM 1634 OD2BASP A 115 30 .401 -3 .501 15 .764 0 .50 20 .69 o
ATOM 1635 C ASP A 115 29 .511 1 .034 14 .682 1 .00 13 .31 c
ATOM 1636 O ASP A 115 30 .488 1 .671 14 .268 1 .00 13 .00 o
ATOM 1637 N ASP A 116 28 .292 1 .098 14 .156 1 .00 12 .95 N ATOM 1639 CA ASP A 116 27,.982 1..996 13..046 1..00 13..15 c
ATOM 1641 CB ASP A 116 26, .618 1, .685 12. .446 1. .00 14. ,36 c
ATOM 1644 CG ASP A 116 26, .506 0. .299 11. .819 1. .00 14. .39 c
ATOM 1645 OD1 ASP A 116 27, .509 -0, .378 11. .592 1. ,00 13. .15 0
ATOM 1646 OD2 ASP A 116 25 .408 -0, .179 11, ,584 1, .00 13, .28 o
ATOM 1647 C ASP A 116 28, .001 3, .437 13, ,581 1. .00 13, .01 c
ATOM 1648 O ASP A 116 28, .529 4, .354 12, ,937 1. .00 13, .99 0
ATOM 1649 N ILE A 117 27, .409 3, .644 14. ,748 1. .00 13, .05 N
ATOM 1651 CA ILE A 117 27 .404 4, .952 15, .339 1. .00 12 .04 C
ATOM 1653 CB ILE A 117 26 .518 4 .961 16, .572 1, .00 12 .89 C
ATOM 1655 CGI ILE A 117 25. .034 4, .744 16, .168 1, .00 17. .41 c
ATOM 1658 CDl ILE A 117 24, .279 3, .948 17, ,085 1. .00 21, .55 c
ATOM 1662 CG2 ILE A 117 26, .715 6, .288 17, ,378 1. .00 15, .14 c
ATOM 1666 C ILE A 117 28 .813 5, .403 15, .623 1. .00 11 .86 c
ATOM 1667 O ILE A 117 29, .195 6, .548 15. .321 1. .00 12, .61 o
ATOM 1668 N ALA A 118 29, .609 4. .532 16. .227 1. ,00 10. ,45 N
ATOM 1670 CA ALA A 118 30, .981 4. .891 16. ,519 1. ,00 11. .56 C
ATOM 1672 CB ALA A 118 31. .649 3. .800 17. ,353 1. ,00 11. .97 C
ATOM 1676 C ALA A 118 31. .786 5, .248 15, .273 1. .00 11, .35 C
ATOM 1677 O ALA A 118 32. .511 6, .241 15, .232 1. .00 10, .56 o
ATOM 1678 N ALA A 119 31, ,597 4, .459 14. .253 1. .00 11, ,09 N
ATOM 1680 CA ALA A 119 32, .298 4. .693 13. .010 1. .00 10. .91 C
ATOM 1682 CB ALA A 119 32, .030 3, .600 12, .104 1. .00 11, ,91 C
ATOM 1686 C ALA A 119 31, .875 6, .029 12, ,430 1. .00 10, .50 C
ATOM 1687 O ALA A 119 32, .721 6. .808 11, .942 1. .00 12, .46 O
ATOM 1688 N ALA A 120 30, .589 6. .342 12, .539 1. .00 11, .95 N
ATOM 1690 CA ALA A 120 30, ,079 7. .579 12. .001 1. ,00 11. ,18 C
ATOM 1692 CB ALA A 120 28 .626 7. .575 12, .034 1. .00 11, .63 C
ATOM 1696 C ALA A 120 30, .643 8, .813 12, ,743 1. .00 11, .63 C
ATOM 1697 O ALA A 120 31, .033 9. .799 12, .104 1. .00 11, .85 O
ATOM 1698 N ILE A 121 30, .708 8. .753 14, .070 1. .00 10, .33 N
ATOM 1700 CA ILE A 121 31, .291 9, .848 14, .892 1. .00 10, .58 C
ATOM 1702 CB ILE A 121 31 .215 9, .481 16, .379 1, ,00 11, .12 C
ATOM 1704 CGI ILE A 121 29, .768 9, .320 16, .750 1. ,00 11, .01 C
ATOM 1707 CDl ILE A 121 29, .566 8, .668 18, ,128 1. .00 11, .36 C
ATOM 1711 CG2 ILE A 121 31, .860 10, ,543 17, ,229 1. .00 12, .83 C
ATOM 1715 C ILE A 121 32 .749 10, .129 14, .510 1. .00 11, .68 C
ATOM 1716 O ILE A 121 33, .158 11, .259 14, .287 1. ,00 12, .00 O
ATOM 1717 N ARG A 122 33, .536 9, .055 14, .448 1. .00 11, .70 N
ATOM 1719 CA ARG A 122 34, .929 9, .171 14, ,046 1. .00 13, .86 C
ATOM 1721 CB ARG A 122 35, .603 7, .810 14, ,114 1. .00 14, .57 C
ATOM 1724 CG ARG A 122 35 .715 7, .320 15 .531 1, .00 14 .77 C
ATOM 1727 CD ARG A 122 36 .384 5. .975 15, .679 1. .00 18. .79 C
ATOM 1730 NE ARG A 122 36, .784 5, .757 17, .048 1, .00 21, .92 N
ATOM 1732 CZ ARG A 122 37, .945 6, .112 17, .577 1. .00 22, .62 C
ATOM 1733 NHl ARG A 122 38 .894 6 .640 16 .838 1, .00 20 .73 N
ATOM 1736 NH2 ARG A 122 38 .178 5 .850 18 .857 1, .00 29 .11 N
ATOM 1739 C ARG A 122 35 .088 9 .760 12 .636 1, .00 14 .45 C
ATOM 1740 O ARG A 122 35 .992 10, .563 12, .389 1. .00 13, .33 O
ATOM 1741 N HIS A 123 34. .198 9, .348 11, .743 1, .00 12, .96 N
ATOM 1743 CA HIS A 123 34 .231 9 .828 10 .385 1, .00 14 .40 C
ATOM 1745 CB HIS A 123 33 .324 9 .001 9 .522 1 .00 14 .46 C
ATOM 1748 CG HIS A 123 33 .390 9 .347 8 .065 1, .00 17 .57 C
ATOM 1749 NDl HIS A 123 3 .358 8 .843 7 .224 1, .00 26 .34 N
ATOM 1751 CEl HIS A 123 34 .183 9 .333 6 .005 1, .00 26 .65 C
ATOM 1753 NE2 HIS A 123 33 .120 10 .115 6 .015 1 .00 23 .02 N
ATOM 1755 CD2 HIS A 123 32 .596 10 .125 7 .299 1, .00 22 .31 C
ATOM 1757 C HIS A 123 33 .913 11 .345 10 .332 1, .00 15 .58 C
ATOM 1758 O HIS A 123 34 .587 12 .095 9 .658 1, .00 14 .13 O
ATOM 1759 N VAL A 124 32 .914 11 .801 11 .081 1 .00 13 .58 N
ATOM 1761 CA VAL A 124 32 .701 13 .233 11, .195 1, .00 13. .94 C
ATOM 1763 CB VAL A 124 31 .583 13 .598 12, .235 1, .00 13, .67 C
ATOM 1765 CGI VAL A 124 31, .476 15, ,111 12, .408 1, .00 11, .98 C ATOM 1769 CG2 VAL A 124 30.,258 13..019 11.,847 1.,00 14.,52 c
ATOM 1773 C VAL A 124 33. ,980 13. .971 11. ,580 1. ,00 12. ,11 c
ATOM 1774 O VAL A 124 34. ,323 14. .973 10. .938 1. .00 13. ,19 O
ATOM 1775 N ALA A 125 34. ,672 13. .495 12. ,604 1. .00 12. ,61 N
ATOM 1777 CA ALA A 125 35, ,875 14. .123 13, .121 1. .00 12. .03 C
ATOM 1779 CB ALA A 125 36. ,351 13. .398 14. ,322 1. ,00 11. .98 C
ATOM 1783 C ALA A 125 36. ,972 14. .158 12. ,062 1. ,00 13. .52 C
ATOM 1784 O ALA A 125 37. ,610 15. .186 11. ,838 1. ,00 13. .20 O
ATOM 1785 N ASP A 126 37. .081 13, .059 11. .312 1. .00 13, .79 N
ATOM 1787 CA ASP A 126 38. .087 12, .980 10, .268 1. .00 15, .94 C
ATOM 1789 CB ASP A 126 38. .180 11, .566 9. .743 1. .00 16, ,11 C
ATOM 1792 CG ASP A 126 38. .895 10, .635 10, .677 1. .00 16, ,70 C
ATOM 1793 OD1 ASP A 126 39. .620 11, .075 11. .580 1. .00 17, ,85 O
ATOM 1794 OD2 ASP A 126 38, .795 9 .393 10, .586 1. .00 17, .09 O
ATOM 1795 C ASP A 126 37. ,736 13. .933 9. .133 1. .00 16. .71 C
ATOM 1796 O ASP A 126 38. ,604 14. ,612 8. .602 1. ,00 17. .22 O
ATOM 1797 N GLU A 127 36. ,465 14. ,027 8. ,798 1. ,00 16. .48 N
ATOM 1799 CA GLU A 127 36. ,033 14. .934 7. ,759 1. ,00 17. .08 C
ATOM 1801 CB GLU A 127 34. .580 14. .723 7. .386 1. .00 17. .14 C
ATOM 1804 CG GLU A 127 34. .319 13. .431 6. .618 1. .00 18. .23 C
ATOM 1807 CD GLU A 127 34. .875 13. .485 5. .205 1. .00 23. .17 C
ATOM 1808 OEl GLU A 127 34. .333 14, .250 4. .412 1. .00 22. .57 O
ATOM 1809 OE GLU A 127 35. .887 12, .809 4, .967 1, .00 25, .80 O
ATOM 1810 C GLU A 127 36. .256 16, .370 8. .204 1. .00 18, .24 C
ATOM 1811 O GLU A 127 36. .634 17, .255 7. .393 1. .00 17, ,10 O
ATOM 1812 N ALA A 128 35. .969 16, .626 9. .465 1. .00 16, ,20 N
ATOM 1814 CA ALA A 128 36. .165 17, .979 10. .000 1. .00 17. .01 C
ATOM 1816 CB ALA A 128 35. .582 18. .100 11, .469 1. .00 18, .11 C
ATOM 1820 C ALA A 128 37, .607 18, .403 9, .959 1. .00 17, .06 C
ATOM 1821 O ALA A 128 37, .923 19, .537 9, .561 1. .00 18, .46 O
ATOM 1822 N SER A 129 38, .496 17, .519 10. .360 1. .00 17, .73 N
ATOM 1824 CA SER A 129 39, .896 17 .869 10, .334 1. .00 19 .04 C
ATOM 1826 CB SER A 129 40, .735 16 .796 10, .996 1. .00 19, .88 C
ATOM 1829 OG SER A 129 40, .289 15 .493 10, .649 1, .00 28, .54 O
ATOM 1831 C SER A 129 40, .367 18 .063 8, .907 1. .00 18, .31 C
ATOM 1832 O SER A 129 41, .158 18 .968 8, .641 1. .00 19, .92 O
ATOM 1833 N ARG A 130 39 .927 17 .209 8 .003 1. .00 17 .50 N
ATOM 1835 CA ARG A 130 40. .418 17 .258 6, .611 1. .00 17 .79 C
ATOM 1837 CB ARG A 130 39, .938 16 .052 5, .802 1, .00 17 .78 C
ATOM 1840 CG ARG A 130 40, .573 15 .989 4, .406 1, .00 17. .70 C
ATOM 1843 CD ARG A 130 40, .048 14 .864 3, .632 1, .00 18, .82 C
ATOM 1846 NE ARG A 130 38 .768 15 .271 3 .064 1 .00 25 .15 N
ATOM 1848 CZ ARG A 130 37 .641 14 .760 3 .393 1 .00 25 .02 C
ATOM 1849 NHl ARG A 130 37 .620 13 .808 4, .337 1. .00 26 .77 N
ATOM 1852 NH2 ARG A 130 36 .524 15 .209 2, .775 1, .00 23 .08 N
ATOM 1855 C ARG A 130 39 .990 18 .543 5 .958 1 .00 19 .20 C
ATOM 1856 O ARG A 130 40 .816 19 .255 5 .352 1 .00 20 .50 O
ATOM 1857 N THR A 131 38 .740 18 .915 6 .155 1 .00 18 .72 N
ATOM 1859 CA THR A 131 38 .127 20 .089 5 .461 1 .00 19 .43 C
ATOM 1861 CB THR A 131 36 .673 19 .866 5 .244 1 .00 20 .43 C
ATOM 1863 OGl THR A 131 35 .973 19 .754 6 .517 1 .00 18 .62 O
ATOM 1865 CG2 THR A 131 36 .421 18 .548 4 .449 1. .00 22 .94 C
ATOM 1869 C THR A 131 38 .262 21 .415 6 .203 1 .00 20 .40 C
ATOM 1870 O THR A 131 37 .906 22 .461 5 .657 1 .00 20 .95 O
ATOM 1871 N GLY A 132 38 .758 21 .356 7 .431 1 .00 18 .57 N
ATOM 1873 CA GLY A 132 38 .841 22 .513 8 .289 1 .00 19 .51 C
ATOM 1876 C GLY A 132 37 .464 23 .129 . 8 .581 1 .00 20 .63 C
ATOM 1877 O GLY A 132 37 .313 24 .336 8 .829 1 .00 23 .12 O
ATOM 1878 N SER A 133 36 .442 22 .287 8 .646 1 .00 18 .55 N
ATOM 1880 CA SER A 133 35 .094 22 .754 8 .904 1 .00 18 .11 C
ATOM 1882 CB SER A 133 34 .080 21 .819 8 .260 1 .00 18 .42 C
ATOM 1885 OG SER A 133 34 .242 21 .666 6 .844 1 .00 21 .03 O
ATOM 1887 C SER A 133 34 .768 22 .836 10 .427 1 .00 16 .26 C ATOM 1888 O SER A 133 35,.348 22..145 11..284 1,.00 15.,54 o
ATOM 1889 N LYS A 134 33, .798 23, .687 10. .720 1. .00 16. ,27 N
ATOM 1891 CA LYS A 134 33. ,275 23. .830 12. ,096 1. .00 15. ,54 C
ATOM 1893 CB LYS A 134 32. ,921 25. .274 12. ,419 1. 00 15. ,50 C
ATOM 1896 CG LYS A 134 34. .154 26. .176 12. ,525 1. .00 18. .18 C
ATOM 1899 CD LYS A 134 33. .819 27, .647 12, ,502 1. ,00 25. .94 C
ATOM 1902 CE LYS A 134 35. ,064 28. .567 12. ,191 1. ,00 30. .57 C
ATOM 1905 NZ LYS A 134 36. ,391 28. .051 12. ,564 1. ,00 34. .14 N
ATOM 1909 C LYS A 134 32. .032 22, .951 12, .094 1. .00 13. .21 C
ATOM 1910 O LYS A 134 31. .121 23, .177 11, .349 1, ,00 14, .18 0
ATOM 1911 N VAL A 135 32. .015 21, .919 12, .921 1, ,00 12. .06 N
ATOM 1913 CA VAL A 135 30. .964 20, ,957 12, .863 1. ,00 12. .02 C
ATOM 1915 CB VAL A 135 31. .487 19, ,632 12. ,321 1. ,00 13. .28 C
ATOM 1917 CGI VAL A 135 30, .363 18, .596 12. .114 1. .00 14. .27 C
ATOM 1921 CG2 VAL A 135 32, .322 19, .867 11. .006 1. .00 14, .42 C
ATOM 1925 C VAL A 135 30, .383 20, .673 14. .241 1. .00 11, .74 C
ATOM 1926 O VAL A 135 31, .097 20, .566 15. .220 1. .00 11, .76 O
ATOM 1927 N VAL A 136 29, .071 20, .604 14. .286 1. .00 11, .28 N
ATOM 1929 CA VAL A 136 28, .389 20 .206 15, .478 1. .00 10. .73 C
ATOM 1931 CB VAL A 136 27, .285 21 .177 15, .819 1. .00 11, .25 C
ATOM 1933 CGI VAL A 136 26, .576 20 .755 17, ,127 1. .00 13, .78 C
ATOM 1937 CG2 VAL A 136 27, .897 22 .594 16, .013 1. .00 12, .65 C
ATOM 1941 C VAL A 136 27 .702 18 .852 15 .159 1, .00 11 .22 C
ATOM 1942 O VAL A 136 26 .973 18 .747 14, .178 1, .00 11 .53 O
ATOM 1943 N ILE A 137 27 .928 17 .850 15, .993 1, .00 9 .65 N
ATOM 1945 CA ILE A 137 27. .255 16, .573 15. ,854 1. .00 10, .61 C
ATOM 1947 CB ILE A 137 28. .113 15, .459 16. .381 1. ,00 9. ,23 C
ATOM 1949 CGI ILE A 137 29. .215 15 .121 15, .395 1. .00 11. .83 C
ATOM 1952 CDl ILE A 137 30. .266 14 .104 15, .930 1. .00 11. .56 C
ATOM 1956 CG2 ILE A 137 27. .238 14, .230 16, .611 1. .00 10, .38 C
ATOM 1960 C ILE A 137 25, .993 16, .610 16. .690 1. .00 10, .44 C
ATOM 1961 O ILE A 137 26, .031 17. .014 17. .869 1. .00 11, .92 O
ATOM 1962 N ASN A 138 24, .899 16 .203 16. .096 1. .00 9 .00 N
ATOM 1964 CA ASN A 138 23, .654 15 .942 16. .764 1. .00 10, .52 C
ATOM 1966 CB ASN A 138 22, .494 16, .601 15. .996 1. .00 8, .55 C
ATOM 1969 CG ASN A 138 21, .146 16, .503 16. .715 1. .00 12, .38 C
ATOM 1970 OD1 ASN A 138 20, .648 17 .515 17. .236 1. .00 10 .74 O
ATOM 1971 ND2 ASN A 138 20, .519 15 .297 16. .722 1. .00 9, .55 N
ATOM 1974 C ASN A 138 23, .376 14 .477 16, .861 1. .00 10, .74 C
ATOM 1975 O ASN A 138 23, .256 13 .799 15, ,833 1. .00 9, .80 O
ATOM 1976 N MET A 139 23, .208 13 .987 18, ,091 1. .00 9, .89 N
ATOM 1978 CA MET A 139 22 .830 12 .585 18 .304 1, .00 10 .28 C
ATOM 1980 CB MET A 139 23 .975 11 .764 18 .906 1, .00 9 .62 C
ATOM 1983 CG MET A 139 24 .984 11 .345 17, .895 1, .00 8 .67 C
ATOM 1986 SD MET A 139 26 .240 10 .206 18, .525 1, .00 11 .34 S
ATOM 1987 CE MET A 139 27 .161 11 .324 19 .556 1 .00 13 .56 C
ATOM 1991 C MET A 139 21 .581 12 .474 19 .162 1 .00 10 .20 C
ATOM 1992 O MET A 139 21 .587 12 .497 20 .413 1 .00 9 .22 O
ATOM 1993 N SER A 140 20 .467 12 .424 18 .458 1 .00 10 .57 N
ATOM 1995 CA SER A 140 19, .166 12 .207 19, .083 1. ,00 10, .10 C
ATOM 1997 CB SER A 140 18 .082 12 .817 18 .201 1. .00 10 .22 C
ATOM 2000 OG SER A 140 18 .142 14 .229 18 .264 1, .00 11 .42 O
ATOM 2002 C SER A 140 18 .959 10 .705 19, .255 1, .00 11 .04 C
ATOM 2003 O SER A 140 18 .006 10 .119 18, .716 1, .00 10 .43 O
ATOM 2004 N LEU A 141 19. .844 10 .063 20, .011 1, .00 10 .62 N
ATOM 2006 CA LEU A 141 19 .890 8 .627 20 .116 1 .00 10 .50 C
ATOM 2008 CB LEU A 141 20 .446 8 .014 18 .833 1. .00 10 .74 C
ATOM 2011 CG LEU A 141 21 .881 8 .483 18 .439 1. .00 11 .49 C
ATOM 2013 CDl LEU A 141 22 .939 7 .897 19 .316 1, .00 11 .20 C
ATOM 2017 CD2 LEU A 141 22 .119 8 .057 17 .001 1 .00 12 .54 C
ATOM 2021 C LEU A 141 20 .748 8 .223 21 .297 1 .00 9 .69 C
ATOM 2022 O LEU A 141 21 .402 9 .060 21 .892 1 .00 9 .38 O
ATOM 2023 N GLY A 142 20 .640 6 .975 21 .689 1 .00 11 .48 N ATOM 2025 CA GLY A 142 21..450 6,.472 22.,789 1..00 10..69 c
ATOM 2028 C GLY A 142 21. .046 5, .146 23. ,335 1. .00 9, .74 c
ATOM 2029 O GLY A 142 20. .283 4, .413 22, ,660 1. .00 12. .20 o
ATOM 2030 N SER A 143 21. .517 4, .850 24. ,562 1. .00 11. .68 N
ATOM 2032 CA SER A 143 21, .200 3 .610 25. .308 1, .00 11, .10 C
ATOM 2034 CB SER A 143 22, .187 2, .489 25. .050 1, .00 11, .52 C
ATOM 2037 OG SER A 143 23, .517 2. .918 25. .316 1. .00 11, .14 O
ATOM 2039 C SER A 143 21, .272 3, .995 26. .777 1, .00 12, .09 C
ATOM 2040 O SER A 143 21, ,941 4, .966 27. .161 1. .00 11, .62 O
ATOM 2041 N SER A 144 20, ,523 3, .292 27. .587 1. .00 11, .90 N
ATOM 2043 CA SER A 144 20, .527 3, .552 28. .992 1. .00 11, .92 C
ATOM 2045 CB SER A 144 19, .513 2. .623 29. .653 1. .00 13, .61 C
ATOM 2048 OG SER A 144 19. .521 2, .848 31. .017 1. .00 18, .36 O
ATOM 2050 C SER A 144 21. .903 3 .362 29. .593 1, .00 13 .78 C
ATOM 2051 O SER A 144 22, .341 4 .161 30, .401 1, .00 14 .05 O
ATOM 2052 N ALA A 145 22, .551 2 .278 29. ,189 1, .00 1 .68 N
ATOM 2054 CA ALA A 145 23, .892 1, .972 29. .678 1, .00 11 .54 C
ATOM 2056 CB ALA A 145 24, .135 0, .503 29. .488 1, .00 12, .43 C
ATOM 2060 C ALA A 145 24 .956 2 .745 28, .886 1. .00 12 .26 C
ATOM 2061 O ALA A 145 24 .814 3. .001 27, .712 1, .00 12 .18 o
ATOM 2062 N LYS A 146 26. .067 3, .066 29. .521 1. .00 10, .71 N
ATOM 2064 CA LYS A 146 27. .184 3, .640 28. .760 1. .00 11, .08 C
ATOM 2066 CB LYS A 146 28, .219 4 .095 29. .754 1, .00 10 .62 C
ATOM 2069 CG LYS A 146 29, .563 4 .535 29. .197 1, .00 13 .37 C
ATOM 2072 CD LYS A 146 30, .506 5 .206 30. .245 1, .00 14 .55 C
ATOM 2075 CE LYS A 146 31, .796 5. .638 29. .643 1. .00 17 .59 C
ATOM 2078 NZ LYS A 146 32, .732 6, .238 30. .665 1. .00 17, .20 N
ATOM 2082 C LYS A 146 27 .767 2 .559 27, .834 1. .00 8 .93 C
ATOM 2083 O LYS A 146 27 .978 1 .383 28, .241 1, .00 11 .42 O
ATOM 2084 N ASP A 147 28, .075 2 .956 26, .621 1, .00 10 .15 N
ATOM 2086 CA ASP A 147 28, .694 2, .129 25, .575 1, .00 10 .08 C
ATOM 2088 CB ASP A 147 27, .843 2 .189 24, .296 1, .00 10 .84 C
ATOM 2091 CG ASP A 147 28, .460 1. .509 23. .117 1, .00 11 .61 C
ATOM 2092 OD1 ASP A 147 29, .701 1 .630 22. .895 1, .00 11 .69 O
ATOM 2093 OD2 ASP A 147 27, .753 0, .879 22. .305 1, .00 10 .96 O
ATOM 2094 C ASP A 147 30, .057 2, .784 25. .353 1. .00 10, .79 C
ATOM 2095 O ASP A 147 30 .163 3 .902 24, .859 1, .00 9 .34 O
ATOM 2096 N SER A 148 31. .104 2 .055 25, .717 1, .00 9 .95 N
ATOM 2098 CA SER A 148 32, .450 2 .588 25, .641 1, .00 11 .43 C
ATOM 2100 CB SER A 148 33, .431 1 .838 26, .533 1, .00 11 .86 C
ATOM 2103 OG SER A 148 33. .164 2 .051 27, .904 1, .00 13 .07 O
ATOM 2105 C SER A 148 32 .999 2 .765 24 .221 1. .00 12 .17 C
ATOM 2106 O SER A 148 33 .958 3 .537 24 .021 1 .00 10 .72 O
ATOM 2107 N LEU A 149 32, .471 1 .997 23, .269 1, .00 9 .59 N
ATOM 2109 CA LEU A 149 32, .936 2 .120 21. .909 1, .00 10 .95 C
ATOM 2111 CB LEU A 149 32 .374 1 .029 21, .030 1 .00 11 .13 C
ATOM 2114 CG LEU A 149 32 .863 1 .087 19, .569 1 .00 13 .82 c
ATOM 2116 CDl LEU A 149 34 .404 0 .961 19, .536 1 .00 14 .72 c
ATOM 2120 CD2 LEU A 149 32 .162 0 .036 18, .699 1 .00 15 .05 c
ATOM 2124 C LEU A 149 32 .474 3 .511 21, .419 1 .00 10 .85 c
ATOM 2125 O LEU A 149 33 .201 4 .268 20 .807 1 .00 10 .58 o
ATOM 2126 N ILE A 150 31 .223 3 .823 21 .649 1 .00 9 .24 N
ATOM 2128 CA ILE A 150 30 .671 5 .127 21 .255 1 .00 9 .06 C
ATOM 2130 CB ILE A 150 29 .145 5 .136 21 .471 1 .00 8 .95 C
ATOM 2132 CGI ILE A 150 28 .499 4 .376 20 .337 1 .00 9 .94 C
ATOM 2135 CDl ILE A 150 27 .038 4 .154 20. .508 1 .00 11 .53 C
ATOM 2139 CG2 ILE A 150 28 .601 6 .553 21, .538 1 .00 11 .45 C
ATOM 2143 C ILE A 150 31 .433 6 .226 22, .052 1 .00 7 .79 c
ATOM 2144 O ILE A 150 31 .793 7 .297 21, .500 1 .00 8 .15 o
ATOM 2145 N ALA A 151 31 .724 5 .956 23 .300 1 .00 8 .34 N
ATOM 2147 CA ALA A 151 32 .396 6 .930 24 .152 1 .00 9 .35 C
ATOM 2149 CB ALA A 151 32 .479 6 .402 25 .577 1 .00 10 .22 C
ATOM 2153 C ALA A 151 33 .796 7 .240 23 .629 1 .00 9 .49 C ATOM 2154 O ALA A 151 34.,215 8..422 23..583 1.,00 9,.45 o
ATOM 2155 N SER A 152 34. ,508 6. .177 23. .181 1. ,00 10, ,40 N
ATOM 2157 CA SER A 152 35. ,823 6. .371 22. ,613 1. ,00 11. ,13 C
ATOM 2159 CB SER A 152 36. ,466 5. .047 22. ,278 1. ,00 10. ,46 C
ATOM 2162 OG SER A 152 37. .628 5. .216 21. .460 1. .00 13. .86 O
ATOM 2164 C SER A 152 35. .737 7, .285 21. .349 1. .00 11. .17 C
ATOM 2165 O SER A 152 36. .585 8. .207 21. .144 1. .00 11. .47 O
ATOM 2166 N ALA A 153 34. .688 7. .103 20. .578 1. ,00 11. .26 N
ATOM 2168 CA ALA A 153 34. .476 7 , .917 19. .358 1. .00 11. .23 C
ATOM 2170 CB ALA A 153 33, .413 7, .295 18. .527 1. .00 12 .05 C
ATOM 2174 C ALA A 153 34, ,143 9, .349 19. .699 1. .00 11 .53 C
ATOM 2175 O ALA A 153 34, ,699 10, .314 19. .103 1. .00 11, .12 O
ATOM 2176 N VAL A 154 33. ,285 9, .529 20. .695 1. .00 9, .67 N
ATOM 2178 CA VAL A 154 32, ,941 10 .878 21, .104 1, .00 9 .77 C
ATOM 2180 CB VAL A 154 31. .908 10. .810 22. .223 1. ,00 10, .26 C
ATOM 2182 CGI VAL A 154 31. .833 12. .136 22. .988 1. ,00 12 , .19 C
ATOM 2186 CG2 VAL A 154 30. .583 10. .402 21. .661 1. ,00 11, .04 C
ATOM 2190 C VAL A 154 34. .229 11. .606 21. ,565 1. ,00 11. .02 C
ATOM 2191 O VAL A 154 34, .449 12, .779 21. .212 1. .00 11, .61 O
ATOM 2192 N ASP A 155 35. .069 10, .954 22. .367 1. .00 10, .47 N
ATOM 2194 CA ASP A 155 36. .309 11, .603 22. .838 1. .00 12, .38 C
ATOM 2196 CB ASP A 155 37. .040 10, .744 23. .859 1. .00 13, .32 C
ATOM 2199 CG ASP A 155 36, .328 10, .668 25. .183 1. .00 19 .66 C
ATOM 2200 OD1 ASP A 155 35, .449 11, .508 25, .455 1. .00 21 .98 O
ATOM 2201 OD2 ASP A 155 36. ,604 9, .813 26, .030 1. .00 21, .73 O
ATOM 2202 C ASP A 155 37. .242 11, .932 21. .674 1. .00 13, .46 C
ATOM 2203 O ASP A 155 37. .928 12. .926 21. .695 1. ,00 12, .65 0
ATOM 2204 N TYR A 156 37, .308 11, .034 20, .694 1. .00 12 .30 N
ATOM 2206 CA TYR A 156 38, .119 11, .263 19, .510 1, .00 11 .91 C
ATOM 2208 CB TYR A 156 37, ,992 10, .073 18, .607 1. .00 12, .85 C
ATOM 2211 CG TYR A 156 38, .753 10, .140 17. .309 1. .00 13, .57 C
ATOM 2212 CDl TYR A 156 40, .154 9, .965 17, .286 1. .00 16. .30 C
ATOM 2214 CEl TYR A 156 40, .822 9 .989 16, .093 1. .00 15 .71 c
ATOM 2216 CZ TYR A 156 40, .136 10, .172 14, .899 1. .00 20 .85 c
ATOM 2217 OH TYR A 156 40, .795 10, .142 13, .677 1. .00 19 .47 0
ATOM 2219 CE2 TYR A 156 38, ,771 10, .352 14, .883 1. .00 14, .75 c
ATOM 2221 CD2 TYR A 156 38, .096 10 .349 16, .111 1, .00 14 .20 c
ATOM 2223 C TYR A 156 37, .653 12 .497 18, .764 1, .00 11 .70 c
ATOM 2224 O TYR A 156 38, .463 13 .343 18, .408 1. .00 12 .40 0
ATOM 2225 N ALA A 157 36, .332 12 .649 18, .630 1. .00 11 .59 N
ATOM 2227 CA ALA A 157 35, .773 13 .776 17, .895 1. .00 10 .92 C
ATOM 2229 CB ALA A 157 34 .319 13 .493 17 .547 1. .00 11 .98 C
ATOM 2233 C ALA A 157 35 .926 15 .058 18 .670 1, .00 11 .69 C
ATOM 2234 O ALA A 157 36 .214 16 .117 18 .072 1, .00 11 .27 o
ATOM 2235 N TYR A 158 35 .740 14 .996 19 .983 1, .00 11 .31 N
ATOM 2237 CA TYR A 158 35 .855 16 .210 20 .809 1 .00 12 .56 C
ATOM 2239 CB TYR A 158 35 .410 15 .940 22 .243 1 .00 12 .28 C
ATOM 2242 CG TYR A 158 35 .147 17 .188 23 .090 1 .00 9 .96 C
ATOM 2243 CDl TYR A 158 34 .015 17 .937 22 .878 1, .00 11 .56 C
ATOM 2245 CEl TYR A 158 33 .754 19 .051 23 .629 1, .00 13 .20 C
ATOM 2247 CZ TYR A 158 34 .635 19 .477 24 .580 1. .00 13 .62 C
ATOM 2248 OH TYR A 158 34 .370 20 .612 25 .295 1 .00 12 .22 O
ATOM 2250 CE2 TYR A 158 35 .813 18 .809 24 .787 1 .00 13 .48 c
ATOM 2252 CD2 TYR A 158 36 .078 17 .647 24 .028 1. .00 12 .56 c
ATOM 2254 C TYR A 158 37 .308 16 .655 20 .783 1. .00 12 .48 c
ATOM 2255 O TYR A 158 37 .591 17 .853 20 .822 1 .00 12 .68 o
ATOM 2256 N GLY A 159 38 .207 15 .683 20 .642 1 .00 12 .38 N
ATOM 2258 CA GLY A 159 39 .628 15 .978 20 .651 1 .00 13 .17 C
ATOM 2261 C GLY A 159 40 .055 16 .611 19 .371 1 .00 13 .46 C
ATOM 2262 O GLY A 159 41 .161 17 .165 19 .297 1 .00 14 .28 O
ATOM 2263 N LYS A 160 39 .238 16 .495 18 .350 1 .00 12 .48 N
ATOM 2265 CA LYS A 160 39 .486 17 .099 17 .035 1 .00 14 .66 C
ATOM 2267 CB LYS A 160 39 .324 16 .046 15 .953 1 .00 15 .36 c ATOM 2270 CG LYS A 160 40..421 14..964 15,.992 1..00 20..16 c
ATOM 2273 CD LYS A 160 40. .057 13, .848 15. .058 1, .00 25. .00 c
ATOM 2276 CE LYS A 160 41. .183 13. .408 14. .161 1. .00 31. .38 c
ATOM 2279 NZ LYS A 160 41. .602 14. .404 13. ,204 1, .00 30. .54 N
ATOM 2283 C LYS A 160 38, .603 18, .344 16. .761 1. .00 14, .03 C
ATOM 2284 O LYS A 160 38, .469 18, .786 15. .621 1. .00 12, .37 O
ATOM 2285 N GLY A 161 38, .076 18, .954 17, .829 1. .00 13 , .97 N
ATOM 2287 CA GLY A 161 37, .363 20, .230 17, .751 1. .00 13 , .11 C
ATOM 2290 C GLY A 161 35 .928 20 .180 17, .281 1. .00 12, .99 C
ATOM 2291 O GLY A 161 35, .434 21 .185 16, .743 1, .00 14, .25 O
ATOM 2292 N VAL A 162 35, .269 19 .020 17, .395 1, .00 11, .44 N
ATOM 2294 CA VAL A 162 33, .858 18, .848 16, ,972 1, .00 10, .32 C
ATOM 2296 CB VAL A 162 33, .621 17, .492 16. .309 1. .00 11, .03 C
ATOM 2298 CGI VAL A 162 32 .146 17 .268 15 .950 1, .00 11 .59 C
ATOM 2302 CG2 VAL A 162 34, .438 17, .378 15. ,034 1. .00 13, .60 C
ATOM 2306 C VAL A 162 32, .991 18, .918 18. .219 1. .00 10, .43 C
ATOM 2307 O VAL A 162 33, .306 18, .259 19. .222 1. .00 11, .54 O
ATOM 2308 N LEU A 163 31, .965 19, .748 18. .217 1. .00 9, .11 N
ATOM 2310 CA LEU A 163 31, .075 19 .817 19, .382 1. .00 10, .40 C
ATOM 2312 CB LEU A 163 30, .278 21 .105 19, .344 1. .00 11, .53 C
ATOM 2315 CG LEU A 163 29, .336 21 .334 20, .515 1. .00 10, ,22 C
ATOM 2317 CDl LEU A 163 30, .163 21, .561 21. .748 1. .00 12, ,89 C
ATOM 2321 CD2 LEU A 163 28 .486 22 .497 20. .248 1, .00 13 .67 C
ATOM 2325 C LEU A 163 30 .118 18 .647 19. .257 1, .00 10 .89 C
ATOM 2326 O LEU A 163 29 .620 18 .367 18, .176 1, .00 13 .34 O
ATOM 2327 N ILE A 164 29 .832 17 .975 20, .347 1. .00 11 ,01 N
ATOM 2329 CA ILE A 164 28, .860 16 .890 20, .383 1. .00 9. ,92 C
ATOM 2331 CB ILE A 164 29 .500 15 .573 20 .908 1. .00 10 .24 C
ATOM 2333 CGI ILE A 164 30 .616 15 .070 19 .976 1, .00 13 .39 C
ATOM 2336 CDl ILE A 164 31, .893 15, .389 20. .491 1. ,00 16, ,69 C
ATOM 2340 CG2 ILE A 164 28, .496 14, .458 20. .907 1. ,00 12, .88 C
ATOM 2344 C ILE A 164 27 .673 17 .259 21. .275 1. .00 10 .29 C
ATOM 2345 O ILE A 164 27, .851 17 .505 22, .479 1, .00 10 .17 O
ATOM 2346 N VAL A 165 26, .489 17 .191 20, ,694 1. .00 9, .19 N
ATOM 2348 CA VAL A 165 25, .257 17, .479 21. ,391 1. .00 9, .03 C
ATOM 2350 CB VAL A 165 24, .576 18, .708 20. .753 1, .00 10, .21 C
ATOM 2352 CGI VAL A 165 23 .300 19 .053 21, .542 1, ,00 9 .33 C
ATOM 2356 CG2 VAL A 165 25 .483 19 .888 20, .715 1, .00 11 .01 C
ATOM 2360 C VAL A 165 24 .360 16 .245 21, .311 1, .00 8 .47 C
ATOM 2361 O VAL A 165 24 .193 15 .681 20, .222 1, .00 10 .40 O
ATOM 2362 N ALA A 166 23 .833 15 .747 22, .452 1. .00 9 .28 N
ATOM 2364 CA ALA A 166 23 .163 14 .469 22 .484 1 .00 8 .57 C
ATOM 2366 CB ALA A 166 24 .104 13 .327 22 .794 1 .00 9 .70 C
ATOM 2370 C ALA A 166 22 .011 14 .454 23 .489 1, .00 9 .40 C
ATOM 2371 O ALA A 166 22 .028 15 .186 24 .476 1. .00 9 .37 O
ATOM 2372 N ALA A 167 21 .000 13 .646 23 .186 1, .00 10 .21 N
ATOM 2374 CA ALA A 167 19 .794 13 .637 23, .965 1, .00 10 .10 C
ATOM 2376 CB ALA A 167 18 .747 12 .726 23, .251 1, .00 11 .66 C
ATOM 2380 C ALA A 167 20 .086 13 .087 25, .329 1, .00 10 .38 C
ATOM 2381 O ALA A 167 20 .787 12 .038 25, .431 1, .00 8 .93 O
ATOM 2382 N ALA A 168 19 .424 13 .572 26 .360 1 .00 10 .36 N
ATOM 2384 CA ALA A 168 19 .623 13 .036 27 .685 1 .00 10 .56 C
ATOM 2386 CB ALA A 168 19 .014 13 .978 28 .698 1 .00 11 .45 C
ATOM 2390 C ALA A 168 19 .026 11 .631 27 .894 1 .00 10 .08 C
ATOM 2391 O ALA A 168 19 .441 10 .860 28 .771 1 .00 10 .97 O
ATOM 2392 N GLY A 169 18 .020 11 .315 27 .108 1 .00 9 .80 N
ATOM 2394 CA GLY A 169 17 .216 10 .125 27 .318 1 .00 9 .88 C
ATOM 2397 C GLY A 169 15 .777 10 .485 27 .780 1 .00 11 .11 C
ATOM 2398 O GLY A 169 15 .483 11 .623 28 .208 1 .00 10 .34 O
ATOM 2399 N ASN A 170 14 .882 9 .492 27 .676 1 .00 11 .54 N
ATOM 2401 CA ASN A 170 13 .483 9 .636 28 .090 1. .00 11 .53 C
ATOM 2403 CB ASN A 170 12 .579 9 .382 26 .872 1. .00 11 .78 C
ATOM 2406 CG ASN A 170 12 .911 10 .285 25 .682 1 .00 13 .65 C ATOM 2407 OD1 ASN A 170 13.358 11.,427 25.856 1.00 14.,26 o
ATOM 2408 ND2 ASN A 170 12. 666 9. .791 24. 465 1. 00 10. ,92 N
ATOM 2411 C ASN A 170 13. 116 8. .658 29. 184 1. 00 12. ,69 C
ATOM 2412 O ASN A 170 12. 046 8. .036 29. 123 1. 00 12. ,66 O
ATOM 2413 N SER A 171 13. 989 8. .483 30. ,170 1. ,00 12. .61 N
ATOM 2415 CA SER A 171 13. 754 7. .487 31. ,223 1. .00 13. .48 C
ATOM 2417 CB SER A 171 15. 025 6. ,692 31. .423 1. .00 14. .78 C
ATOM 2420 OG SER A 171 15. 277 5. ,967 30. ,233 1. .00 13. .11 O
ATOM 2422 C SER A 171 13. ,308 8. .147 32, .538 1, .00 13. .22 C
ATOM 2423 O SER A 171 13. ,429 7. .526 33. .583 1. .00 14, .40 O
ATOM 2424 N GLY A 172 12. ,811 9. .371 32. ,496 1. ,00 14, .32 N
ATOM 2426 CA GLY A 172 12. ,428 10. .098 33. ,710 1. ,00 14. .13 C
ATOM 2429 C GLY A 172 11. ,127 9. .606 34. ,292 1. ,00 15. .66 C
ATOM 2430 O GLY A 172 10. .473 8. .814 33. .614 1. .00 15, .22 O
ATOM 2431 N SER A 173 10. ,681 10. .134 35. .424 1. .00 14, .94 N
ATOM 2433 CA SER A 173 11. ,269 11. .278 36. .134 1. .00 16, .57 C
ATOM 2435 CB SER A 173 10. ,144 12. .174 36. ,639 1. .00 17, .91 C
ATOM 2438 OG SER A 173 9. ,384 11. .435 37. .607 1. ,00 18, .04 O
ATOM 2440 C SER A 173 12. .196 10. .908 37. .265 1. .00 16. .47 C
ATOM 2441 O SER A 173 12. .751 11. .790 37. .970 1. .00 15, .55 O
ATOM 2442 N GLY A 174 12. ,476 9. .615 37. .359 1. .00 15, .06 N
ATOM 2444 CA GLY A 174 13. ,318 9. .075 38. .400 1. .00 16, .35 C
ATOM 2447 C GLY A 174 14, .715 9. .629 38. .233 1. .00 17 .64 C
ATOM 2448 O GLY A 174 15, .159 9, .906 37. .086 1. .00 17, .46 O
ATOM 2449 N SER A 175 15. .404 9, .827 39. .351 1. .00 17 .08 N
ATOM 2451 CA SER A 175 16. .752 10, .404 39. .336 1. .00 18, .22 C
ATOM 2453 CB SER A 175 17. .129 10, .794 40. .759 1. .00 19, .80 C
ATOM 2456 OG SER A 175 16, .121 11, .654 41, .308 1, .00 21 .20 O
ATOM 2458 C SER A 175 17, .783 9, .457 38, .777 1, .00 16 .85 C
ATOM 2459 O SER A 175 17, .638 8, .238 38, .884 1. .00 15 .37 O
ATOM 2460 N ASN A 176 18, .838 10, .010 38, .168 1. .00 16 .57 N
ATOM 2462 CA ASN A 176 19 .966 9 .230 37, .675 1, .00 15 .32 C
ATOM 2464 CB ASN A 176 20, .679 8 .475 38, .817 1, .00 17 .33 C
ATOM 2467 CG ASN A 176 22, .174 8 .352 38, .565 1, .00 20 .64 C
ATOM 2468 OD1 ASN A 176 22, .676 9, .003 37, .649 1, .00 19 .65 O
ATOM 2469 ND2 ASN A 176 22, .881 7, .508 39, .336 1, .00 23 .68 N
ATOM 2472 C ASN A 176 19 .634 8 .250 36. .592 1 .00 15 .22 C
ATOM 2473 O ASN A 176 20 .208 7 .146 36, .528 1, .00 16 .86 O
ATOM 2474 N THR A 177 18 .718 8 .639 35, .723 1, .00 14 .53 N
ATOM 2476 CA THR A 177 18, .299 7 .815 34, .612 1, .00 14 .30 C
ATOM 2478 CB THR A 177 16, .768 7, .831 34, .488 1, .00 13 .61 C
ATOM 2480 OGl THR A 177 16 .255 9 .161 34 .632 1 .00 12 .76 O
ATOM 2482 CG2 THR A 177 16 .053 7 .001 35 .629 1 .00 14 .89 C
ATOM 2486 C THR A 177 18 .907 8 .265 33. .267 1, .00 13 .62 C
ATOM 2487 O THR A 177 18 .555 7 .704 32, .213 1, .00 14 .34 O
ATOM 2488 N ILE A 178 19 .736 9 .305 33 .324 1 .00 12 .33 N
ATOM 2490 CA ILE A 178 20 .436 9 .809 32 .125 1 .00 10 .85 C
ATOM 2492 CB ILE A 178 21 .473 10 .886 32, .543 1 .00 11 .28 C
ATOM 2494 CGI ILE A 178 22 .118 11 .576 31. .337 1. .00 11 .95 C
ATOM 2497 CDl ILE A 178 22 .981 12 .833 31, ,722 1, .00 12 .82 C
ATOM 2501 CG2 ILE A 178 22 .550 10 .300 33 .406 1 .00 11 .10 C
ATOM 2505 C ILE A 178 21 .057 8 .663 31 .350 1 .00 10 .78 C
ATOM 2506 O ILE A 178 21 .582 7 .715 31 .950 1 .00 10 .96 O
ATOM 2507 N GLY A 179 20 .973 8 .706 30 .032 1 .00 9 .07 N
ATOM 2509 CA GLY A 179 21 .658 7 .732 29 .200 1 .00 10 .43 C
ATOM 2512 C GLY A 179 22 .842 8 .334 28 .433 1 .00 10 .17 C
ATOM 2513 O GLY A 179 23 .302 9 .423 28 .716 1 .00 9 .05 O
ATOM 2514 N PHE A 180 23 .302 7 .579 27 .474 1 .00 10 .42 N
ATOM 2516 CA PHE A 180 24 .566 7 .766 26 .771 1 .00 9 .90 C
ATOM 2518 CB PHE A 180 25 .597 6 .710 27 .248 1 .00 11 .20 C
ATOM 2521 CG PHE A 180 25 .926 6 .868 28 .691 1 .00 10 .40 C
ATOM 2522 CDl PHE A 180 25 .089 6 .304 29 .673 1 .00 12 .43 C
ATOM 2524 CEl PHE A 180 25 .346 6 .539 31 .013 1 .00 13 .80 C ATOM 2526 CZ PHE A 180 26..377 7..353 31.,379 1,.00 14,.77 c
ATOM 2528 CE2 PHE A 180 27. .195 7. .936 30. ,428 1. .00 14, .15 c
ATOM 2530 CD2 PHE A 180 26. .951 7, .710 29. ,086 1, .00 12. .50 c
ATOM 2532 C PHE A 180 24. ,307 7. .663 25. ,268 1, ,00 10. .49 c
ATOM 2533 O PHE A 180 23. .545 6. .804 24. .833 1. .00 11, .55 0
ATOM 2534 N PRO A 181 25. .023 8, .448 24. .458 1. .00 9, .50 N
ATOM 2535 CA PRO A 181 26. .196 9, .246 24. ,890 1. .00 9, .13 C
ATOM 2537 CB PRO A 181 26. .937 9. .496 23. ,524 1. .00 9, .24 C
ATOM 2540 CG PRO A 181 25, .855 9, .591 22. .649 1, .00 11, .31 C
ATOM 2543 CD PRO A 181 24, ,874 8, .496 23. .002 1, .00 10, .03 c
ATOM 2546 C PRO A 181 25, .983 10. .570 25. .610 1. .00 10, .79 c
ATOM 2547 O PRO A 181 26, ,959 11, .251 25. .986 1. .00 9, .76 0
ATOM 2548 N GLY A 182 24. .743 11, .019 25. .752 1. .00 10, .07 N
ATOM 2550 CA GLY A 182 24, .480 12 .260 26. .450 1, .00 10 .91 C
ATOM 2553 C GLY A 182 25, .260 12, .460 27, .748 1, .00 10 .65 C
ATOM 2554 O GLY A 182 25, .843 13, .532 27, .983 1. ,00 10 .24 O
ATOM 2555 N GLY A 183 25, .246 11, .400 28. .570 1. .00 10, .23 N
ATOM 2557 CA GLY A 183 25. .860 11, .370 29. .888 1. ,00 11, .34 C
ATOM 2560 C GLY A 183 27, .370 11 .393 29, .929 1, .00 11 .75 C
ATOM 2561 O GLY A 183 27, .957 11 .394 31, .025 1, .00 10 .33 O
ATOM 2562 N LEU A 184 28, .007 11, .351 28, .761 1. .00 11 .62 N
ATOM 2564 CA LEU A 184 29, .474 11, .436 28. .718 1, .00 11, .77 C
ATOM 2566 CB LEU A 184 30, .022 11 .038 27, .369 1. .00 10 .76 C
ATOM 2569 CG LEU A 184 29, .612 9 .640 26, .946 1. .00 13 .28 c
ATOM 2571 CDl LEU A 184 29. .958 9 .447 25, .484 1, .00 13 .01 c
ATOM 2575 CD2 LEU A 184 30, .306 8, .563 27, .839 1, .00 15 .87 c
ATOM 2579 C LEU A 184 29, .966 12, .827 29. .034 1, .00 11, .00 c
ATOM 2580 O LEU A 184 29 .321 13 .827 28, .682 1, .00 10 .77 0
ATOM 2581 N VAL A 185 31, .180 12 .922 29, .584 1, .00 11 .17 N
ATOM 2583 CA VAL A 185 31. .673 14, .225 29, ,979 1, .00 10 .77 C
ATOM 2585 CB VAL A 185 32. .994 14, .115 30. .811 1. .00 13, .00 C
ATOM 2587 CGI VAL A 185 33, .978 13 .569 30, ,013 1, .00 16 .83 C
ATOM 2591 CG2 VAL A 185 33, .500 15 .504 31, .144 1, .00 16 .03 c
ATOM 2595 C VAL A 185 31, .868 15, .172 28, .842 1, .00 9 .95 c
ATOM 2596 O VAL A 185 31, .683 16, .370 28. .972 1, .00 10, .97 0
ATOM 2597 N ASN A 186 32, .181 14, .614 27. .684 1, .00 10, .43 N
ATOM 2599 CA ASN A 186 32 .483 15 .376 26, .517 1, .00 11 .79 C
ATOM 2601 CB ASN A 186 33 .763 14 .865 25, .836 1, .00 13 .55 C
ATOM 2604 CG ASN A 186 35, .029 15 .152 26, .660 1, .00 14 .82 c
ATOM 2605 OD1 ASN A 186 35, .093 16 .110 27, .370 1, .00 19 .19 0
ATOM 2606 ND2 ASN A 186 36, .021 14 .326 26, .517 1, .00 21 .64 N
ATOM 2609 C ASN A 186 31 .305 15 .525 25 .536 1 .00 11 .85 c
ATOM 2610 O ASN A 186 31 .485 15 .915 24, .384 1 .00 11 .39 0
ATOM 2611 N ALA A 187 30 .108 15 .138 25, .977 1, .00 10 .51 N
ATOM 2613 CA ALA A 187 28, .904 15 .382 25, .179 1, .00 11 .08 C
ATOM 2615 CB ALA A 187 28 .189 14 .150 24 .848 1. .00 11 .19 C
ATOM 2619 C ALA A 187 27 .984 16 .317 25 .975 1 .00 10 .66 C
ATOM 2620 O ALA A 187 27 .878 16 .147 27 .186 1 .00 11 .05 O
ATOM 2621 N VAL A 188 27 .318 17 .248 25 .288 1 .00 9 .26 N
ATOM 2623 CA VAL A 188 26, .326 18 .148 25, .895 1, .00 9 .21 C
ATOM 2625 CB VAL A 188 26 .120 19 .418 25 .046 1 .00 9 .41 C
ATOM 2627 CGI VAL A 188 25 .035 20 .249 25 .661 1 .00 7 .57 C
ATOM 2631 CG2 VAL A 188 27 .448 20 .164 24 .893 1 .00 10 .01 C
ATOM 2635 C VAL A 188 24 .996 17 .346 25, .984 1 .00 9 .78 C
ATOM 2636 O VAL A 188 24 .349 17 .137 24 .959 1 .00 10 .45 O
ATOM 2637 N ALA A 189 24 .572 16 .989 27 .200 1 .00 8 .67 N
ATOM 2639 CA ALA A 189 23 .325 16 .236 27 .430 1 .00 9 .35 C
ATOM 2641 CB ALA A 189 23 .379 15 .510 28 .763 1 .00 10 .27 C
ATOM 2645 C ALA A 189 22. .197 17 .214 27 .451 1 .00 8 .43 C
ATOM 2646 O ALA A 189 22 .179 18 .183 28 .238 1 .00 9 .35 O
ATOM 2647 N VAL A 190 21 .182 16 .948 26 .651 1 .00 9 .14 N
ATOM 2649 CA VAL A 190 20 .084 17 .882 26 .554 1 .00 8 .32 C
ATOM 2651 CB VAL A 190 19 .843 18 .296 25 .119 1 .00 8 .58 C ATOM 2653 CGI VAL A 190 18,.731 19,.309 25..052 1,.00 11.50 c
ATOM 2657 CG2 VAL A 190 21, .084 18, .873 24. .482 1, .00 9, .20 c
ATOM 2661 C VAL A 190 18, .791 17, .317 27. .087 1. .00 9, .51 c
ATOM 2662 O VAL A 190 18, .340 16, .256 26. ,625 1. .00 9, .51 0
ATOM 2663 N ALA A 191 18, .236 17 .973 28. .093 1, .00 9 .50 N
ATOM 2665 CA ALA A 191 16, .934 17, .559 28. .685 1, .00 10 .18 C
ATOM 2667 CB ALA A 191 16, .868 18, .057 30. .134 1, .00 8 .69 C
ATOM 2671 C ALA A 191 15, .800 18, .184 27, ,900 1. .00 10, .24 C
ATOM 2672 O ALA A 191 16, .007 19, .182 27. .249 1. .00 10 .28 O
ATOM 2673 N ALA A 192 14, .570 17, .659 28. .021 1. .00 11 .22 N
ATOM 2675 CA ALA A 192 13, .428 18, .171 27. .272 1. .00 11, .79 C
ATOM 2677 CB ALA A 192 12, .593 17, .012 26, ,728 1. .00 13, .73 C
ATOM 2681 C ALA A 192 12. .499 19. .027 28. ,134 1. .00 11, .54 C
ATOM 2682 O ALA A 192 12, .048 18, .549 29. .179 1. .00 11 .30 O
ATOM 2683 N LEU A 193 12 .222 20 .238 27. .673 1, .00 11 .58 N
ATOM 2685 CA LEU A 193 11 .194 21 .103 28. .258 1, .00 11 .55 C
ATOM 2687 CB LEU A 193 11, .519 22, .561 28. .037 1, .00 12 .06 c
ATOM 2690 CG LEU A 193 12, .844 23, .095 28. .613 1, .00 10. .40 c
ATOM 2692 CDl LEU A 193 13 .137 24 .484 28, .211 1, .00 9 .32 c
ATOM 2696 CD2 LEU A 193 12 .752 22 .903 30. .081 1, .00 13 .29 c
ATOM 2700 C LEU A 193 9, .852 20, .802 27. .577 1, .00 14. .05 c
ATOM 2701 O LEU A 193 9. .814 20, .460 26. .414 1, .00 13 .73 o
ATOM 2702 N GLU A 194 8 .755 21 .004 28, .305 1, .00 13 .56 N
ATOM 2704 CA GLU A 194 7 .422 21 .049 27, .647 1, .00 13 .63 C
ATOM 2706 CB GLU A 194 6, .472 20, .125 28. .359 1, .00 12 .74 C
ATOM 2709 CG GLU A 194 6, .320 20, .410 29. .837 1, ,00 16. .62 C
ATOM 2712 CD GLU A 194 5, .490 19, .404 30. .603 1. .00 19, .10 C
ATOM 2713 OEl GLU A 194 5 .288 18 .280 30. .118 1, .00 20 .53 0
ATOM 2714 OE2 GLU A 194 5, .153 19, .744 31. .765 1, ,00 17 .20 0
ATOM 2715 C GLU A 194 6 .934 22 .460 27. ,699 1, .00 14 .48 c
ATOM 2716 O GLU A 194 7 .502 23 .275 28. .431 1, .00 13 .32 0
ATOM 2717 N ASN A 195 5 .862 22 .778 26, .954 1, .00 14 .74 N
ATOM 2719 CA ASN A 195 5 .416 24 .169 26, .857 1, .00 15 .00 C
ATOM 2721 CB ASN A 195 4 .773 24 .467 25, ,479 1, .00 16 .14 C
ATOM 2724 CG ASN A 195 4 .612 25 .970 25. .198 1, .00 16 .46 c
ATOM 2725 OD1 ASN A 195 5, .236 26, .803 25. .855 1, .00 14 .84 o
ATOM 2726 ND2 ASN A 195 3 .775 26 .318 24, .208 1, .00 13 .91 N
ATOM 2729 C ASN A 195 4 .439 24 .440 27. .945 1, .00 16 .03 C
ATOM 2730 O ASN A 195 3, .256 24, .692 27. .662 1. .00 15 .98 O
ATOM 2731 N VAL A 196 4, .904 24, .381 29. .170 1, ,00 16 .13 N
ATOM 2733 CA VAL A 196 4, .106 24, .623 30. ,344 1. .00 16, ,89 C
ATOM 2735 CB VAL A 196 3 .739 23 .348 31. .019 1, .00 18 .38 C
ATOM 2737 CGI VAL A 196 3, .058 23 , .613 32. .326 1. ,00 20 .19 C
ATOM 2741 CG2 VAL A 196 2, .922 22, .415 30. .070 1. .00 18, .32 C
ATOM 2745 C VAL A 196 4, ,991 25, .380 31. ,307 1. .00 17, .79 c
ATOM 2746 O VAL A 196 6 .215 25 .147 31. .344 1 .00 17 .15 O
ATOM 2747 N GLN A 197 4 .410 26 .305 32, .055 1 .00 17 .34 N
ATOM 2749 CA GLN A 197 5 .171 27 .048 33, .060 1, .00 16 .33 C
ATOM 2751 CB GLN A 197 4 .838 28 .518 33, ,012 1, .00 16 .74 C
ATOM 2754 CG GLN A 197 4 .987 29 .169 31. ,720 1. .00 16 .89 C
ATOM 2757 CD GLN A 197 6 .455 29 .357 31, ,343 1 .00 18 .48 C
ATOM 2758 OEl GLN A 197 7 .216 30 .009 32, ,096 1, .00 14 .66 O
ATOM 2759 NE2 GLN A 197 6 .850 28 .769 30, .223 1, .00 15 .37 N
ATOM 2762 C GLN A 197 4 .907 26, .538 34. .467 1, .00 17 .79 C
ATOM 2763 O GLN A 197 3. .778 26. .114 34. .825 1. .00 18 .50 O
ATOM 2764 N GLN A 198 5 .977 26 .409 35. .232 1, .00 17 .07 N
ATOM 2766 CA GLN A 198 5 .879 25 .977 36. .627 1, .00 17 .39 C
ATOM 2768 CB GLN A 198 5 .865 24, .485 36. .778 1. .00 17 .84 C
ATOM 2771 CG GLN A 198 5, .744 24, .058 38. .164 1. .00 18 .38 c
ATOM 2774 CD GLN A 198 5 .797 22 .558 38. ,413 1, .00 25 .24 c
ATOM 2775 OEl GLN A 198 6 .612 21 .813 37, .815 1. .00 26 .03 0
ATOM 2776 NE2 GLN A 198 4 .927 22 .090 39, .323 1 .00 29 .00 N
ATOM 2779 C GLN A 198 6 .998 26 .623 37, .362 1, .00 17 .51 C ATOM 2780 O GLN A 198 8.,156 26.,681 36.,904 1.,00 16.,23 o
ATOM 2781 N ASN A 199 6. 655 27. ,147 38. ,520 1. ,00 18. ,31 N
ATOM 2783 CA ASN A 199 7. 612 27. ,890 39. ,321 1. 00 19. ,42 C
ATOM 2785 CB ASN A 199 8. 676 26. ,953 39. 915 1. 00 19. ,53 C
ATOM 2788 CG ASN A 199 8. .107 25. .949 40. .861 1. .00 24. .01 C
ATOM 2789 OD1 ASN A 199 7. .226 26. .254 41. ,691 1. .00 23. .71 0
ATOM 2790 ND2 ASN A 199 8. ,598 24, .738 40. ,769 1. ,00 25. .09 N
ATOM 2793 C ASN A 199 8. ,285 29, ,018 38. ,592 1. ,00 20. .51 C
ATOM 2794 O ASN A 199 9. .491 29 , .356 38. .863 1. .00 20, .07 O
ATOM 2795 N GLY A 200 7. .533 29. .653 37. .712 1. .00 19, .65 N
ATOM 2797 CA GLY A 200 8. .005 30. .820 36. .991 1. .00 20, .16 C
ATOM 2800 C GLY A 200 8. ,883 30. .609 35. .774 1. .00 19, .26 C
ATOM 2801 O GLY A 200 9. ,347 31. .577 35. .177 1. .00 18. .58 O
ATOM 2802 N THR A 201 9, .091 29. .348 35. .384 1. .00 17, .29 N
ATOM 2804 CA THR A 201 9. .876 29, .045 34. .196 1. .00 16, .82 C
ATOM 2806 CB THR A 201 11. .327 28, ,563 34. .596 1. .00 16, .41 C
ATOM 2808 OGl THR A 201 11. .309 27. ,246 35. .174 1. .00 17, .63 O
ATOM 2810 CG2 THR A 201 11. ,954 29. .437 35. .622 1. .00 17, .27 C
ATOM 2814 C THR A 201 9, .248 27, .935 33. .389 1, .00 15 .50 C
ATOM 2815 O THR A 201 8, .267 27, .356 33. .817 1. .00 14, .83 O
ATOM 2816 N TYR A 202 9. .845 27, .608 32. .230 1. .00 15, .66 N
ATOM 2818 CA TYR A 202 9. .469 26, ,407 31. .571 1. .00 14, .51 C
ATOM 2820 CB TYR A 202 10, .308 26, .183 30, .300 1, .00 15 .38 C
ATOM 2823 CG TYR A 202 9, .853 27, .053 29, .177 1, .00 12 .00 C
ATOM 2824 CDl TYR A 202 8, .775 26, .682 28, .359 1. .00 12 .96 C
ATOM 2826 CEl TYR A 202 8, ,344 27, .510 27, .364 1. .00 13 .55 C
ATOM 2828 CZ TYR A 202 8. ,995 28, .682 27. .157 1. .00 12, .70 C
ATOM 2829 OH TYR A 202 8, .586 29 .559 26, .172 1, .00 14 .68 O
ATOM 2831 CE2 TYR A 202 10, .027 29, .063 27, .961 1, .00 14 .77 C
ATOM 2833 CD2 TYR A 202 10, .441 28, .245 28, .957 1, .00 13 .26 C
ATOM 2835 C TYR A 202 9, .637 25. .229 32, .488 1, .00 14 .45 C
ATOM 2836 O TYR A 202 10. .442 25 .231 33 .415 1 .00 14 .03 O
ATOM 2837 N ARG A 203 8, .894 24 .168 32 .206 1, .00 14 .53 N
ATOM 2839 CA ARG A 203 8, .939 22. .971 32, .988 1, .00 13 .25 C
ATOM 2841 CB ARG A 203 7, .454 22. .543 33, .262 1, .00 14 .52 C
ATOM 2844 CG ARG A 203 7, .315 21, .347 34, .102 1, .00 14 .19 C
ATOM 2847 CD ARG A 203 5 .795 21 .083 34 .523 1 .00 15 .99 C
ATOM 2850 NE ARG A 203 5 .730 20 .106 35. .572 1 .00 17 .45 N
ATOM 2852 CZ ARG A 203 5 .729 18 .806 35 .402 1, .00 17 .33 C
ATOM 2853 NHl ARG A 203 5, .762 18 .306 34, .191 1, .00 15 .77 N
ATOM 2856 NH2 ARG A 203 5, .741 17 .996 36, .447 1, .00 18 .53 N
ATOM 2859 C ARG A 203 9 .595 21 .805 32 .251 1 .00 12 .75 C
ATOM 2860 O ARG A 203 9 .285 21 .570 31 .076 1 .00 12 .82 O
ATOM 2861 N VAL A 204 10 .505 21 .090 32 .907 1 .00 12 .18 N
ATOM 2863 CA VAL A 204 11 .043 19 .877 32, .378 1, .00 11 .32 C
ATOM 2865 CB VAL A 204 12 .141 19 .338 33 .296 1 .00 11 .73 C
ATOM 2867 CGI VAL A 204 12 .765 18 .035 32 .735 1 .00 11 .27 C
ATOM 2871 CG2 VAL A 204 13 .229 20 .373 33 .437 1. .00 12 .71 C
ATOM 2875 C VAL A 204 9 .925 18 .831 32 .221 1 .00 12 .28 C
ATOM 2876 O VAL A 204 9 .172 18 .549 33 .185 1 .00 12 .08 O
ATOM 2877 N ALA A 205 9 .855 18 .166 31 .063 1 .00 12 .11 N
ATOM 2879 CA ALA A 205 8 .882 17 .061 30 .935 1 .00 12 .63 C
ATOM 2881 CB ALA A 205 8 .725 16 .612 29 .473 1 .00 14 .20 C
ATOM 2885 C ALA A 205 9 .205 15 .894 31 .835 1 .00 14 .44 C
ATOM 2886 O ALA A 205 10 .338 15 .546 32 .067 1 .00 12 .72 O
ATOM 2887 N ASP A 206 8 .160 15 .251 32 .353 1 .00 12 .46 N
ATOM 2889 CA ASP A 206 8 .351 14 .138 33 .226 1 .00 14 .42 C
ATOM 2891 CB ASP A 206 7 .015 13 .579 33 .660 1 .00 15 .15 C
ATOM 2894 CG ASP A 206 6 .273 14 .456 34 .620 1 .00 20 .28 C
ATOM 2895 OD1 ASP A 206 6 .717 15 .552 35 .015 1 .00 16 .78 O
ATOM 2896 OD2 ASP A 206 5 .164 14 .025 35 .032 1 .00 21 .17 O
ATOM 2897 C ASP A 206 9 .161 13 .020 32 .556 1 .00 13 .89 C
ATOM 2898 O ASP A 206 9 .920 12 .348 33 .229 1 .00 15 .04 O ATOM 2899 N PHE A 207 9,.016 12.821 31,.246 1..00 11..78 N
ATOM 2901 CA PHE A 207 9, .710 11 .721 30, .612 1. .00 13. .96 C
ATOM 2903 CB PHE A 207 9, .163 11, .360 29. ,213 1. .00 13. .87 C
ATOM 2906 CG PHE A 207 9. .290 12, .439 28. ,191 1. .00 14, .05 C
ATOM 2907 CDl PHE A 207 10, .521 12 .704 27. .630 1. ,00 13 .46 C
ATOM 2909 CEl PHE A 207 10, .677 13 .677 26. .709 1. ,00 14, .99 C
ATOM 2911 CZ PHE A 207 9, .577 14 .463 26. .305 1. .00 12, .88 C
ATOM 2913 CE2 PHE A 207 8, .325 14, .173 26. .841 1. .00 15, .57 C
ATOM 2915 CD2 PHE A 207 8, .199 13 .191 27. .787 1. .00 14 .48 C
ATOM 2917 C PHE A 207 11, .220 11 .917 30. .546 1. .00 12 .81 C
ATOM 2918 O PHE A 207 11, .950 10 .945 30. .339 1. .00 12, .43 o
ATOM 2919 N SER A 208 11, .670 13 .163 30. ,626 1. .00 13, .24 N
ATOM 2921 CA SER A 208 13, .099 13, .459 30. ,389 1. .00 12, .69 C
ATOM 2923 CB SER A 208 13 .277 14 .980 30. .333 1. .00 13 .69 C
ATOM 2926 OG SER A 208 14 .593 15 .399 30, .016 1, .00 11 .00 O
ATOM 2928 C SER A 208 13 .997 12 .799 31, .432 1, .00 11 .85 C
ATOM 2929 O SER A 208 13 .726 12 .841 32. .612 1, .00 12 .56 O
ATOM 2930 N SER A 209 15 .095 12 .168 31. .001 1, .00 11 .34 N
ATOM 2932 CA SER A 209 15 .961 11 .503 31. .941 1 .00 12 .26 C
ATOM 2934 CB SER A 209 17 .003 10 .655 31. .240 1, .00 10 .68 C
ATOM 2937 OG SER A 209 16 .442 9 .566 30, .515 1, .00 11 .25 O
ATOM 2939 C SER A 209 16 .666 12 .506 32, .852 1, .00 11 .42 C
ATOM 2940 O SER A 209 17 .108 13 .568 32, .420 1. .00 12 .21 O
ATOM 2941 N ARG A 210 16 .797 12 .128 34, .107 1, .00 13 .04 N
ATOM 2943 CA ARG A 210 17 .480 12 .973 35, .089 1, .00 12 .02 C
ATOM 2945 CB ARG A 210 16 .783 12 .925 36. .439 1, .00 11 .62 C
ATOM 2948 CG ARG A 210 15, .644 13 .914 36, .659 1. ,00 10 .95 C
ATOM 2951 CD ARG A 210 14 .531 13 .936 35. .593 1, .00 12 .61 C
ATOM 2954 NE ARG A 210 13 .496 14 .936 35, .948 1, .00 13 .59 N
ATOM 2956 CZ ARG A 210 12 .450 15 .250 35. .214 1, .00 14 .23 C
ATOM 2957 NHl ARG A 210 12, .267 14 .655 34. .035 1. .00 14 .26 N
ATOM 2960 NH2 ARG A 210 11 .550 16 .168 35, .652 1, .00 13 .18 N
ATOM 2963 C ARG A 210 18 .906 12 .544 35, .328 1, .00 11 .40 C
ATOM 2964 O ARG A 210 19 .201 11 .401 35. .281 1, .00 12 .42 O
ATOM 2965 N GLY A 211 19, .756 13 .520 35. .625 1. .00 13, .31 N
ATOM 2967 CA GLY A 211 21, .140 13 .289 35, .977 1, .00 11. .79 C
ATOM 2970 C GLY A 211 21 .263 12 .757 37, .395 1, .00 13 .79 C
ATOM 2971 O GLY A 211 20 .286 12 .467 38. .054 1, .00 13 .33 O
ATOM 2972 N ASN A 212 22. .508 12 .644 37. .831 1. .00 12 .68 N
ATOM 2974 CA ASN A 212 22, .852 1 .132 39. .139 1. .00 13 .86 C
ATOM 2976 CB ASN A 212 24, .300 11 .647 39. .062 1. .00 14, .05 C
ATOM 2979 CG ASN A 212 24 .801 11 .076 40. .380 1, .00 15 .77 C
ATOM 2980 OD1 ASN A 212 24 .034 10 .961 41. .330 1. .00 20 .22 O
ATOM 2981 ND2 ASN A 212 26. .057 10 .638 40. .402 1. .00 21 .14 N
ATOM 2984 C ASN A 212 22, .711 13 .289 40. .154 1. .00 13 .72 C
ATOM 2985 O ASN A 212 23 .466 14 .254 40, .141 1, .00 13 .40 O
ATOM 2986 N PRO A 213 21 .803 13 .173 41. .121 1. .00 14 .98 N
ATOM 2987 CA PRO A 213 21 .672 14 .248 42. .125 1, .00 16 .29 C
ATOM 2989 CB PRO A 213 20, .586 13 .722 43. .065 1. ,00 17 .58 C
ATOM 2992 CG PRO A 213 19, .803 12 .803 42. .224 1. ,00 16 .96 C
ATOM 2995 CD PRO A 213 20 .863 12 .072 41. .386 1, ,00 15 .73 C
ATOM 2998 C PRO A 213 22 .966 14 .577 42. .864 1. ,00 16 .46 C
ATOM 2999 O PRO A 213 23. .194 15 .766 43. .089 1. ,00 18 .16 O
ATOM 3000 N ALA A 214 23, .809 13 .573 43. .084 1. .00 17 .81 N
ATOM 3002 CA ALA A 214 25 .058 13 .706 43. .843 1, .00 18 .79 C
ATOM 3004 CB ALA A 214 25 .746 12 .336 44. .004 1. .00 19 .37 C
ATOM 3008 C ALA A 214 26, .022 14 .653 43. .188 1. .00 18 .92 C
ATOM 3009 O ALA A 214 26, .872 15 .225 43. .890 1. .00 17 .86 O
ATOM 3010 N THR A 215 25, .899 14 .879 41. .869 1. .00 17. .03 N
ATOM 3012 CA THR A 215 26 .868 15 .751 41. .205 1, .00 16 .66 C
ATOM 3014 CB THR A 215 27 .741 14 .942 40, .212 1 .00 18 .38 C
ATOM 3016 OGl THR A 215 26 .907 14 .218 39, .271 1, .00 15 .43 O
ATOM 3018 CG2 THR A 215 28 .532 13 .928 40, .970 1, .00 18 .05 C ATOM 3022 C THR A 215 26.278 16.952 40.479 1, 00 16.40 C
ATOM 3023 O THR A 215 26.955 17.636 39.745 1, 00 16.41 O
ATOM 3024 N ALA A 216 25.035 17.244 40.773 1, 00 14.91 N
ATOM 3026 CA ALA A 216 24.408 18.446 40.249 1.00 16.14 C
ATOM 3028 CB ALA A 216 22.944 18.182 39.938 1.00 16.49 C
ATOM 3032 C ALA A 216 24.479 19.548 41.271 1 00 17.73 C
ATOM 3033 O ALA A 216 24.240 19.279 42.445 1 00 21.27 O
ATOM 3034 N GLY A 217 24.699 20.763 40.840 1 00 16.83 N
ATOM 3036 CA GLY A 217 24.701 21.920 41.725 1 00 17.35 C
ATOM 3039 C GLY A 217 25.994 22.704 41.603 1 00 18.14 C
ATOM 3040 O GLY A 217 26.068 23.906 41.983 1 00 19.91 O
ATOM 3041 N ASP A 218 27.007 22.103 41.001 1 00 17.40 N
ATOM 3043 CA ASP A 218 28.294 22.767 40.941 1, 00 16.68 C
ATOM 3045 CB ASP A 218 29.346 21.732 41.259 1 00 17.36 C
ATOM 3048 CG ASP A 218 29.393 20.585 40.244 1 00 20.24 C
ATOM 3049 OD1 ASP A 218 28.520 20.437 39.333 1 00 17.05 O
ATOM 3050 OD2 ASP A 218 30.286 19.734 40.356 1 00 20.78 O
ATOM 3051 C ASP A 218 28.648 23.526 39.664 1 00 15.65 C
ATOM 3052 O ASP A 218 29.638 24.231 39.613 1 00 15.75 O
ATOM 3053 N TYR A 219 27.756 23.522 38.689 1 00 15.41 N
ATOM 3055 CA TYR A 219 28.003 24.099 37.380 1 00 13.99 C
ATOM 3057 CB TYR A 219 27.987 25.607 37.463 1 00 14.94 C
ATOM 3060 CG TYR A 219 26.611 26.197 37.674 1 00 15.83 C
ATOM 3061 CDl TYR A 219 25.642 26.086 36.702 1 00 12.98 C
ATOM 3063 CEl TYR A 219 24.385 26.648 36.871 1 00 12.57 C
ATOM 3065 CZ TYR A 219 24.125 27.301 38.052 1 00 18.74 C
ATOM 3066 OH TYR A 219 22.947 27.921 38.286 1 00 18.11 O
ATOM 3068 CE2 TYR A 219 25.090 27.438 39.021 1 00 19.74 C
ATOM 3070 CD2 TYR A 219 26.309 26.869 38.843 1 00 19.78 C
ATOM 3072 C TYR A 219 29.346 23.584 36.756 1 00 14.17 C
ATOM 3073 O TYR A 219 29.978 24.283 35.984 1 00 14.67 o
ATOM 3074 N ILE A 220 29.676 22.330 37.031 1 00 13.56 N
ATOM 3076 CA ILE A 220 30.775 21.624 36.414 1 00 15.58 C
ATOM 3078 CB ILE A 220 31.961 21.438 37.355 1 00 15.45 C
ATOM 3080 CGI ILE A 220 32.502 22.809 37.761 1 00 20.96 C
ATOM 3083 CDl ILE A 220 33.397 22.711 39.015 1 00 23.15 C
ATOM 3087 CG2 ILE A 220 33.087 20.625 36.671 1 00 18.47 C
ATOM 3091 C ILE A 220 30.227 20.278 35.956 1 00 13.00 c
ATOM 3092 O ILE A 220 29.633 19.505 36.691 1 00 12.94 o
ATOM 3093 N ILE A 221 30.482 19.995 34.684 1 00 15.43 N
ATOM 3095 CA ILE A 221 29.934 18.782 34.088 1 00 13.93 C
ATOM 3097 CB ILE A 221 29.793 18.960 32.541 1 00 12.79 C
ATOM 3099 CGI ILE A 221 28.733 19.979 32.164 1 00 12.82 C
ATOM 3102 CDl ILE A 221 27.361 19.711 32.673 1 00 12.70 c
ATOM 3106 CG2 ILE A 221 29.513 17.651 31.889 1 00 14.76 c
ATOM 3110 C ILE A 221 30.836 17.584 34.406 1 00 14.63 c
ATOM 3111 O ILE A 221 32.059 17.583 34.006 1 00 14.50 0
ATOM 3112 N GLN A 222 30.246 16.602 35.097 1 00 13.31 N
ATOM 3114 CA GLN A 222 30.786 15.267 35.285 1 00 14.28 C
ATOM 3116 CB GLN A 222 30.772 14.858 36.759 1 00 16.33 C
ATOM 3119 CG GLN A 222 31.775 15.669 37.602 1 00 18.83 C
ATOM 3122 CD GLN A 222 31.204 16.936 38.223 1 00 21.01 C
ATOM 3123 OEl GLN A 222 29.998 17.058 38.469 1 00 22.83 O
ATOM 3124 NE2 GLN A 222 32.089 17.874 38.517 1 00 23.39 N
ATOM 3127 C GLN A 222 29.935 14.289 34.460 1 00 14.17 C
ATOM 3128 O GLN A 222 28.921 14.664 33.874 1 00 12.09 O
ATOM 3129 N GLU A 223 30.438 13.079 34.313 1 00 13.56 N
ATOM 3131 CA GLU A 223 29.646 12.052 33.675 1 00 14.32 C
ATOM 3133 CB GLU A 223 30.455 10.767 33.682 1 00 14.62 C
ATOM 3136 CG GLU A 223 29.787 9.618 32.984 1 00 15.34 C
ATOM 3139 CD GLU A 223 30.759 8.474 32.706 1 00 16.41 C
ATOM 3140 OEl GLU A 223 31.400 8.407 31.648 1 00 19.83 O
ATOM 3141 OE2 GLU A 223 30.808 7.645 33.564 1 00 19.76 O ATOM 3142 C GLU A 223 28,.315 11..911 34..451 1..00 12..77 c
ATOM 3143 O GLU A 223 28, ,294 11, .981 35, .679 1. .00 13, .97 o
ATOM 3144 N ARG A 224 27, .210 11, .785 33, .700 1. .00 11, .67 N
ATOM 3146 CA ARG A 224 25, .834 11, .585 34. .178 1. .00 11, .39 C
ATOM 3148 CB ARG A 224 25, .730 10 .549 35. .305 1. .00 12 .52 C
ATOM 3151 CG ARG A 224 26, .269 9 .156 34. .960 1. .00 16, .20 C
ATOM 3154 CD ARG A 224 25, .988 8 .116 36. .088 1, .00 19, .39 C
ATOM 3157 NE ARG A 224 24, .589 7, .737 36. .052 1. .00 19, .97 N
ATOM 3159 CZ ARG A 224 24, .123 6 .785 35. .271 1, .00 23 .20 C
ATOM 3160 NHl ARG A 224 24, .939 6 .070 34. .516 1. .00 22 .67 N
ATOM 3163 NH2 ARG A 224 22, .835 6, .530 35. .234 1. .00 20, .20 N
ATOM 3166 C ARG A 224 25, ,199 12, .934 34. .600 1. .00 11, .27 C
ATOM 3167 O ARG A 224 24, ,137 12, .954 35. .247 1. .00 12, .76 O
ATOM 3168 N ASP A 225 25, ,779 14 .065 34. .167 1, .00 10 .64 N
ATOM 3170 CA ASP A 225 25 .141 15 .322 34, .440 1 .00 11 .57 C
ATOM 3172 CB ASP A 225 26 .137 16 .421 34, .763 1 .00 12 .18 C
ATOM 3175 CG ASP A 225 26 .783 16 .290 36, .115 1, .00 11 .47 C
ATOM 3176 OD1 ASP A 225 26 .396 15 .431 36. .930 1, .00 12 .63 O
ATOM 3177 OD2 ASP A 225 27 .738 17 .024 36, .366 1 .00 12 .98 O
ATOM 3178 C ASP A 225 24 .386 15 .833 33, .187 1 .00 11 .72 C
ATOM 3179 O ASP A 225 24 .880 15 .761 32, .028 1, .00 10 .65 O
ATOM 3180 N ILE A 226 23, .195 16 .366 33. ,428 1, .00 10 .89 N
ATOM 3182 CA ILE A 226 22 .449 17 .089 32, .399 1 .00 10 .95 C
ATOM 3184 CB ILE A 226 20 .988 17 .379 32, .826 1, .00 10 .97 C
ATOM 3186 CGI ILE A 226 20, .296 16 .066 33, .224 1, .00 8 .65 C
ATOM 3189 CDl ILE A 226 20, .064 15 .192 32. .004 1, .00 12, .39 C
ATOM 3193 CG2 ILE A 226 20, .260 18, .217 31. .828 1, .00 12, .31 C
ATOM 3197 C ILE A 226 23 .117 18 .428 32, .195 1, .00 8 .74 C
ATOM 3198 O ILE A 226 23 .561 19 .076 33, ,123 1, .00 10 .81 O
ATOM 3199 N GLU A 227 23, .254 18 .849 30. .971 1, .00 9 .24 N
ATOM 3201 CA GLU A 227 23, .902 20 .150 30. .715 1, .00 9, .53 C
ATOM 3203 CB GLU A 227 24 .905 19 .979 29. .553 1, .00 8 .94 c
ATOM 3206 CG GLU A 227 25 .869 21 .131 29. .345 1, .00 9 .79 c
ATOM 3209 CD GLU A 227 27. .217 20 .634 28. .789 1, .00 11 .40 c
ATOM 3210 OEl GLU A 227 27, .527 19 .431 28. .857 1, .00 8, .56 0
ATOM 3211 OE2 GLU A 227 27, .953 21, .441 28. .255 1. .00 11, .05 0
ATOM 3212 C GLU A 227 22 .936 21 .331 30. .450 1, .00 9 .10 c
ATOM 3213 O GLU A 227 23, .053 22 .402 31. .074 1, .00 10 .35 o
ATOM 3214 N VAL A 228 21. .984 21 .152 29. ,553 1. .00 8 .61 N
ATOM 3216 CA VAL A 228 21, .094 22 .222 29. .176 1, .00 7. .54 c
ATOM 3218 CB VAL A 228 21, .567 23, .010 27. .946 1. .00 5, .96 c
ATOM 3220 CGI VAL A 228 22 .834 23 .726 28, .263 1, .00 8 .28 c
ATOM 3224 CG2 VAL A 228 21 .680 22 .127 26, .740 1, .00 8 .08 c
ATOM 3228 C VAL A 228 19 .727 21 .575 28, .904 1, .00 7 .83 c
ATOM 3229 O VAL A 228 19, .663 20 .362 28, .747 1, .00 9 .43 0
ATOM 3230 N SER A 229 18 .716 22 .426 28, .809 1 .00 10 .13 N
ATOM 3232 CA SER A 229 17 .369 22 .024 28, .520 1 .00 9 .88 C
ATOM 3234 CB SER A 229 16 .457 22 .371 29, .674 1. .00 10 .86 C
ATOM 3237 OG SER A 229 16 .811 21 .670 30, ,827 1, .00 11 .90 o
ATOM 3239 C SER A 229 16 .865 22 .806 27. ,305 1. .00 11 .07 c
ATOM 3240 O SER A 229 17 .290 23 .949 27, .065 1 .00 11 .59 0
ATOM 3241 N ALA A 230 15 .964 22 .167 26, .536 1, .00 10 .22 N
ATOM 3243 CA ALA A 230 15 .337 22 .824 25, .379 1, .00 9 .98 C
ATOM 3245 CB ALA A 230 16 .245 22 .786 24. .202 1, .00 11 .98 C
ATOM 3249 C ALA A 230 14 .001 22 .181 25, .011 1 .00 10 .40 C
ATOM 3250 O ALA A 230 13 .639 21 .143 25, .539 1 .00 11 .74 O
ATOM 3251 N PRO A 231 13 .241 22 .873 24, .162 1, .00 12 .52 N
ATOM 3252 CA PRO A 231 11 .915 22 .369 23, .794 1, .00 11 .45 C
ATOM 3254 CB PRO A 231 11 .440 23 .308 22, .676 1, .00 12 .82 C
ATOM 3257 CG PRO A 231 12 .076 24 .609 23 .005 1 .00 12 .47 C
ATOM 3260 CD PRO A 231 13 .518 24 .184 23 .540 1 .00 12 .90 C
ATOM 3263 C PRO A 231 11 .967 20 .969 23 .241 1 .00 10 .90 C
ATOM 3264 O PRO A 231 12 .689 20 .713 22 .238 1 .00 11 .39 O ATOM 3265 N GLY A 232 11..194 20..071 23,,863 1..00 11..69 N
ATOM 3267 CA GLY A 232 11. .218 18. .675 23, ,478 1. .00 12. .12 C
ATOM 3270 C GLY A 232 9. .857 17. .972 23, ,431 1. .00 14. .63 C
ATOM 3271 O GLY A 232 9. .814 16. .852 22. ,976 1. .00 17. .38 O
ATOM 3272 N ALA A 233 8. .775 18, .641 23. .807 1, .00 13, .80 N
ATOM 3274 CA ALA A 233 7. .441 17, .975 23. .841 1. .00 15, .69 C
ATOM 3276 CB ALA A 233 6. .812 18, .002 25. .228 1. .00 15, .50 C
ATOM 3280 C ALA A 233 6. .565 18. .649 22. .794 1. .00 15, .70 C
ATOM 3281 O ALA A 233 6. .479 19, .892 22, .725 1, .00 15 .71 O
ATOM 3282 N SER A 234 6. .008 17, .837 21, .901 1, .00 15 .70 N
ATOM 3284 CA SER A 234 5, .123 18, ,323 20, .820 1, .00 17 .34 C
ATOM 3286 CB SER A 234 3. .816 18, .866 21, .396 1, .00 19, .34 C
ATOM 3289 OG SER A 234 3. ,151 17, .862 22. .071 1. .00 24, .09 O
ATOM 3291 C SER A 234 5, .746 19 .302 19, .869 1, .00 16 .08 c
ATOM 3292 O SER A 234 5. ,311 20, .419 19. .727 1. .00 16, .37 o
ATOM 3293 N VAL A 235 6. ,816 18, ,859 19. .244 1. .00 13, .70 N
ATOM 3295 CA VAL A 235 7. .597 19. .607 18. .309 1. .00 13, .30 C
ATOM 3297 CB VAL A 235 9. .124 19. .358 18. .536 1. .00 11. .29 C
ATOM 3299 CGI VAL A 235 9. .948 19. .994 17. .533 1. .00 11, .79 C
ATOM 3303 CG2 VAL A 235 9. .475 19, .925 19. .919 1. .00 13, .78 C
ATOM 3307 C VAL A 235 7. .284 19, .242 16. .876 1. .00 14, .46 C
ATOM 3308 O VAL A 235 7. .529 18, .141 16. .413 1. .00 15, .04 O
ATOM 3309 N GLU A 236 6. .773 20, ,208 16. .151 1. .00 15 .81 N
ATOM 3311 CA GLU A 236 6. .539 20, .075 14, .717 1. .00 15 .96 C
ATOM 3313 CB GLU A 236 5, .419 21, .059 14, .323 1. .00 16, .03 C
ATOM 3316 CG GLU A 236 5. .033 21, .028 12. .863 1. .00 18, .99 C
ATOM 3319 CD GLU A 236 3. .833 21, ,939 12. .549 1. .00 22, .94 C
ATOM 3320 OEl GLU A 236 3, .422 22 .715 13. .457 1. .00 20 .60 O
ATOM 3321 OE2 GLU A 236 3, .255 21, .772 11. .420 1. .00 20 .10 O
ATOM 3322 C GLU A 236 7. .751 20, .349 13, .881 1, .00 16 .01 C
ATOM 3323 O GLU A 236 8. .534 21, .272 14, .139 1. .00 17, .39 O
ATOM 3324 N SER A 237 8. .023 19 .462 12, .905 1, .00 14 .14 N
ATOM 3326 CA SER A 237 9, .105 19 .655 12, .025 1, .00 14 .47 C
ATOM 3328 CB SER A 237 10. .410 19. .109 12, .632 1, .00 11 .88 C
ATOM 3331 OG SER A 237 11. .513 19, .504 11, .921 1, .00 11 .55 O
ATOM 3333 C SER A 237 8. .819 18, .900 10, .715 1, .00 14 .97 C
ATOM 3334 O SER A 237 7. .699 18 .345 10, .543 1 .00 16 .60 O
ATOM 3335 N THR A 238 9. .838 18 .892 9, .886 1, .00 15 .64 N
ATOM 3337 CA THR A 238 9, .851 18 .230 8, .581 1, .00 17 .00 C
ATOM 3339 CB THR A 238 11, .090 18 .559 7, .844 1, .00 18 .10 C
ATOM 3341 OGl THR A 238 12, .267 18, .454 8, .677 1, .00 16 .55 O
ATOM 3343 CG2 THR A 238 11 .152 19 .996 7 .339 1 .00 16 .33 C
ATOM 3347 C THR A 238 9 .752 16 .703 8 .759 1 .00 19 .28 C
ATOM 3348 O THR A 238 10 .213 16 .169 9 .768 1 .00 18 .16 O
ATOM 3349 N TRP A 239 9, .203 16 .032 7, .739 1, .00 19 .48 N
ATOM 3351 CA TRP A 239 8 .933 14 .587 7 .765 1 .00 18 .83 C
ATOM 3353 CB TRP A 239 7 .503 14 .322 8 .122 1 .00 19 .25 C
ATOM 3356 CG TRP A 239 7 .182 12 .954 8 .642 1, .00 19 .71 C
ATOM 3357 CDl TRP A 239 6 .343 1 .009 8 .085 1 .00 23 .51 C
ATOM 3359 NEl TRP A 239 6 .263 10 .907 8, .899 1 .00 22 .49 N
ATOM 3361 CE2 TRP A 239 7 .081 11 .114 9 .985 1 .00 21 .32 C
ATOM 3362 CD2 TRP A 239 7 .651 12 .400 9 .853 1 .00 20 .74 C
ATOM 3363 CE3 TRP A 239 8 .529 12 .864 10 .851 1 .00 18 .32 C
ATOM 3365 CZ3 TRP A 239 8 .751 12 .055 11 .962 1 .00 21 .08 C
ATOM 3367 CH2 TRP A 239 8 .166 10 .788 12 .061 1 .00 20 .53 C
ATOM 3369 CZ2 TRP A 239 7 .349 10 .289 11 .079 1 .00 22 .21 C
ATOM 3371 C TRP A 239 9 .325 13 .931 6 .461 1 .00 17 .68 C
ATOM 3372 O TRP A 239 9 .417 14 .577 5 .423 1 .00 19 .48 O
ATOM 3373 N TYR A 240 9 .679 12 .642 6 .550 1 .00 17 .80 N
ATOM 3375 CA TYR A 240 10 .332 11 .947 5 .485 1 .00 17 .76 C
ATOM 3377 CB TYR A 240 10 .862 10 .568 5 .938 1 .00 20 .08 C
ATOM 3380 CG TYR A 240 9 .864 9 .469 6 .036 1 .00 17 .92 C
ATOM 3381 CDl TYR A 240 8 .997 9 .367 7 .097 1 .00 20 .96 C ATOM 3383 CEl TYR A 240 8.113 8.,325 7.179 1.00 22.,25 c
ATOM 3385 CZ TYR A 240 8. 150 7. ,317 6. 171 1. 00 27. ,67 c
ATOM 3386 OH TYR A 240 7. 269 6. ,247 6. 207 1. 00 28. ,63 o
ATOM 3388 CΞ2 TYR A 240 9. 034 7. ,397 5. 159 1. 00 25. ,36 c
ATOM 3390 CD2 TYR A 240 9. ,884 8. .442 5. ,084 1. ,00 24. ,19 c
ATOM 3392 C TYR A 240 9. ,453 11. .746 4. 253 1. ,00 19. .14 c
ATOM 3393 O TYR A 240 9. 992 11. ,532 3. 199 1. ,00 19, .72 0
ATOM 3394 N THR A 241 8. 172 11. ,873 4. 401 1. ,00 21, .32 N
ATOM 3396 CA THR A 241 7. ,309 11. .777 3. ,214 1. .00 24, ,31 C
ATOM 3398 CB THR A 241 6. ,015 11. .129 3. ,549 1, ,00 24, .08 C
ATOM 3400 OGl THR A 241 5. ,443 11. .711 4. ,720 1. ,00 25, ,38 o
ATOM 3402 CG2 THR A 241 6. ,238 9. .670 3. ,924 1. ,00 28, .14 C
ATOM 3406 C THR A 241 7. ,020 13. .113 2. ,587 1. ,00 26. ,01 c
ATOM 3407 O THR A 241 6. .175 13. .200 1. .682 1. .00 27, ,85 o
ATOM 3408 N GLY A 242 7. .684 14. .159 3. .041 1. ,00 26. ,13 N
ATOM 3410 CA GLY A 242 7. ,445 15. .480 2. .484 1. ,00 26. .65 C
ATOM 3413 C GLY A 242 6, ,544 16. .381 3. .303 1. ,00 25. ,41 C
ATOM 3414 O GLY A 242 6. ,524 17. .583 3. 068 1. .00 29. ,27 O
ATOM 3415 N GLY A 243 5. ,786 15, .891 4. ,246 1. .00 23. .67 N
ATOM 3417 CA GLY A 243 5. ,002 16. .882 4. ,959 1. .00 23. ,78 C
ATOM 3420 C GLY A 243 5. ,719 17. .300 6. ,239 1. ,00 21, ,57 C
ATOM 3421 O GLY A 243 6. ,944 17. .532 6. ,218 1. ,00 20, ,45 O
ATOM 3422 N TYR A 244 4, .939 17, .380 7. .296 1. .00 22, ,18 N
ATOM 3424 CA TYR A 244 5. .404 17, .745 8. .651 1. .00 21, ,26 C
ATOM 3426 CB TYR A 244 4. .991 19, .201 8. .963 1. .00 20, .92 C
ATOM 3429 CG TYR A 244 5. .467 20, .078 7. .877 1. .00 20, .48 C
ATOM 3430 CDl TYR A 244 4. .696 20, .312 6. ,749 1. .00 20, .98 C
ATOM 3432 CEl TYR A 244 5, .167 21 .082 5, .733 1, .00 21 .20 C
ATOM 3434 CZ TYR A 244 6. .396 21, .604 5. .785 1, .00 20 .81 C
ATOM 3435 OH TYR A 244 6, .852 22, .332 4. .745 1. .00 23 .64 O
ATOM 3437 CE2 TYR A 244 7. .201 21, .392 6. .929 1. .00 17 .10 C
ATOM 3439 CD2 TYR A 244 6, .714 20 .660 7. .920 1. .00 14 .54 C
ATOM 3441 C TYR A 244 4, .789 16 .816 9, .621 1, .00 21 .28 c
ATOM 3442 O TYR A 244 3, .778 16 .224 9, ,353 1, .00 23 .43 0
ATOM 3443 N ASN A 245 5, .381 16. .692 10. .800 1. .00 19 .64 N
ATOM 3445 CA ASN A 245 4, .866 15 .855 11. .825 1. .00 18 .70 C
ATOM 3447 CB ASN A 245 5 .322 14 .418 11, .590 1. .00 18 .51 C
ATOM 3450 CG ASN A 245 4 .644 13 .438 12, .450 1. .00 18 .67 C
ATOM 3451 OD1 ASN A 245 3 .509 13 .611 12, .925 1. .00 21 .26 O
ATOM 3452 ND2 ASN A 245 5 .350 12 .323 12, .689 1, .00 22 .00 N
ATOM 3455 C ASN A 245 5 .304 16 .401 13, .154 1, .00 16 .91 C
ATOM 3456 O ASN A 245 6 .289 17 .194 13 .212 1 .00 17 .80 O
ATOM 3457 N THR A 246 4 .533 16 .035 14 .141 1 .00 17 .22 N
ATOM 3459 CA THR A 246 4 .696 16 .445 15, .504 1 .00 17 .23 C
ATOM 3461 CB THR A 246 3 .454 17 .242 15, .971 1 .00 17 .67 C
ATOM 3463 OGl THR A 246 3 .368 18 .458 15 .223 1 .00 18 .24 O
ATOM 3465 CG2 THR A 246 3 .534 17 .710 17 .400 1. .00 20 .37 C
ATOM 3469 C THR A 246 4 .993 15 .244 16 .374 1 .00 17 .33 C
ATOM 3470 O THR A 246 4 .123 14 .398 16 .664 1 .00 16 .60 o
ATOM 3471 N ILE A 247 6 .221 15 .237 16 .902 1 .00 16 .92 N
ATOM 3473 CA ILE A 247 6 .655 14 .201 17 .813 1 .00 16 .77 C
ATOM 3475 CB ILE A 247 7 .536 13 .106 17 .091 1 .00 16 .85 C
ATOM 3477 CGI ILE A 247 8 .722 13 .699 16 .372 1 .00 16 .22 C
ATOM 3480 CDl ILE A 247 9 .721 12 .674 15 .892 1 .00 14 .19 C
ATOM 3484 CG2 ILE A 247 6 .620 12 .272 16 .155 1 .00 18 .78 C
ATOM 3488 C ILE A 247 7 .394 14 .839 19 .045 1 .00 14 .93 C
ATOM 3489 O ILE A 247 7 .628 16 .050 19 .098 1 .00 15 .83 O
ATOM 3490 N SER A 248 7 .659 14 .020 20 .030 1 .00 14 .46 N
ATOM 3492 CA SER A 248 8 .268 14 .443 21 .285 1 .00 14 .38 C
ATOM 3494 CB SER A 248 7 .262 14 .230 22 .426 1 .00 14 .68 C
ATOM 3497 OG SER A 248 6 .080 14 .988 22 .271 1 .00 16 .96 O
ATOM 3499 C SER A 248 9 .470 13 .594 21 .672 1 .00 13 .40 C
ATOM 3500 O SER A 248 9 .626 12 .448 21 .227 1. .00 12 .06 O ATOM 3501 N GLY A 249 10..306 14..151 22.,557 1.,00 11..90 N
ATOM 3503 CA GLY A 249 11. .402 13. .420 23. ,140 1. ,00 12. .25 C
ATOM 3506 C GLY A 249 12. .615 14. ,294 23. ,393 1. ,00 11. .45 C
ATOM 3507 O GLY A 249 12. .672 15. .399 22. ,898 1. ,00 12. .12 O
ATOM 3508 N THR A 250 13, .607 13. .759 24. .103 1. .00 10, .99 N
ATOM 3510 CA THR A 250 14. .898 14. .476 24. ,200 1. .00 9, .84 C
ATOM 3512 CB THR A 250 15. .835 13. ,937 25. ,257 1. .00 9. .57 C
ATOM 3514 OGl THR A 250 15. .885 12. ,543 25. ,195 1. ,00 10. .83 O
ATOM 3516 CG2 THR A 250 15. .260 14, .258 26. .612 1. .00 11, .12 C
ATOM 3520 C THR A 250 15. .506 14. .531 22. .800 1. .00 9, .75 c
ATOM 3521 O THR A 250 16. .312 15. .393 22. ,517 1. .00 11, .31 o
ATOM 3522 N SER A 251 15. .101 13. .616 21. ,938 1. .00 9. .71 N
ATOM 3524 CA SER A 251 15. .396 13. .711 20. ,513 1. ,00 10. .29 C
ATOM 3526 CB SER A 251 14, .647 12, .660 19. .691 1. .00 10, .44 C
ATOM 3529 OG SER A 251 15 .278 11, .374 19. .720 1, .00 11 .64 O
ATOM 3531 C SER A 251 15, .132 15, .058 19, .850 1, .00 10 .60 C
ATOM 3532 O SER A 251 15, .871 15, .475 18, .968 1. .00 12, .05 O
ATOM 3533 N MET A 252 14, .023 15, .698 20. .246 1. .00 11, .35 N
ATOM 3535 CA MET A 252 13 .598 16 .937 19, .649 1, .00 12 .05 C
ATOM 3537 CB MET A 252 12 .081 17, .000 19, .803 1. .00 10 .74 C
ATOM 3540 CG MET A 252 11. .275 16, .163 18, .811 1. .00 16 .05 C
ATOM 3543 SD MET A 252 11. .445 14, .393 18, .855 1, .00 12 .67 S
ATOM 3544 CE MET A 252 12 .360 1 .105 17 .508 1. .00 14 .70 C
ATOM 3548 C MET A 252 14 .248 18 .146 20 .363 1. .00 11 .00 C
ATOM 3549 O MET A 252 14 .372 19 .200 19, .805 1, .00 10 .97 O
ATOM 3550 N ALA A 253 14 .615 17, .978 21, .628 1, .00 11 .44 N
ATOM 3552 CA ALA A 253 15 .300 19, .023 22, .361 1. .00 9 .30 C
ATOM 3554 CB ALA A 253 15 .282 18 .677 23 .852 1. .00 8 .63 C
ATOM 3558 C ALA A 253 16 .733 19 .234 21 .842 1, .00 9 .29 C
ATOM 3559 O ALA A 253 17 .173 20 .367 21, .599 1, .00 10 .72 O
ATOM 3560 N THR A 254 17 .388 18, .107 21, .578 1, .00 9 .20 N
ATOM 3562 CA THR A 254 18 .776 18 .047 21 .127 1 .00 9 .63 C
ATOM 3564 CB THR A 254 19 .131 16 .592 20 .838 1. .00 11 .67 C
ATOM 3566 OGl THR A 254 19 .001 15 .823 22 .036 1, .00 9 .34 O
ATOM 3568 CG2 THR A 254 20 .504 16 .467 20, .434 1, .00 9 .85 C
ATOM 3572 C THR A 254 19 .041 18, .957 19, .927 1, .00 9 .01 C
ATOM 3573 O THR A 254 19 .932 19 .782 19 .958 1 .00 9 .02 O
ATOM 3574 N PRO A 255 18 .255 18 .848 18 .856 1 .00 10 .43 N
ATOM 3575 CA PRO A 255 18 .491 19 .725 17 .711 1, .00 9 .72 C
ATOM 3577 CB PRO A 255 17 .607 19 .149 16 .621 1, .00 11 .04 C
ATOM 3580 CG PRO A 255 16 .511 18, .430 17, .368 1, .00 11 .23 C
ATOM 3583 CD PRO A 255 17 .253 17 .817 18 .535 1 .00 11 .38 C
ATOM 3586 C PRO A 255 18 .195 21 .185 17 .941 1, .00 10 .12 C
ATOM 3587 O PRO A 255 18 .724 22 .018 17 .171 1, .00 9 .83 O
ATOM 3588 N HIS A 256 17 .398 21 .535 18, .943 1, .00 10 .53 N
ATOM 3590 CA HIS A 256 17 .233 22 .947 19 .219 1 .00 10 .79 C
ATOM 3592 CB HIS A 256 16 .192 23 .258 20 .298 1, .00 11 .66 C
ATOM 3595 CG HIS A 256 14 .748 23 .136 19 .820 1, .00 12 .48 C
ATOM 3596 NDl HIS A 256 14 .086 21 .930 19 .732 1, .00 11 .32 N
ATOM 3598 CEl HIS A 256 12 .849 22 .144 19, .296 1, .00 14 .50 C
ATOM 3600 NE2 HIS A 256 12 .709 23 .434 19 .040 1 .00 12 .79 N
ATOM 3602 CD2 HIS A 256 13 .889 24 .074 19 .348 1 .00 12 .20 C
ATOM 3604 C HIS A 256 18 .572 23 .451 19 .658 1. .00 11 .12 C
ATOM 3605 O HIS A 256 18 .977 24 .554 19 .302 1, .00 12 .50 O
ATOM 3606 N VAL A 257 19 .264 22 .660 20 .474 1 .00 10 .13 N
ATOM 3608 CA VAL A 257 20 .558 23 .078 20 .969 1 .00 9 .71 C
ATOM 3610 CB VAL A 257 20 .966 22 .264 22 .219 1 .00 10 .12 C
ATOM 3612 CGI VAL A 257 22 .407 22 .589 22 .653 1 .00 10 .84 C
ATOM 3616 CG2 VAL A 257 19 .951 22 .451 23, .324 1, .00 11 .41 C
ATOM 3620 C VAL A 257 21 .632 22 .993 19 .871 1 .00 10 .99 C
ATOM 3621 O VAL A 257 22 .483 23 .871 19 .782 1 .00 11 .55 O
ATOM 3622 N ALA A 258 21 .664 21 .933 19 .070 1 .00 10 .95 N
ATOM 3624 CA ALA A 258 22 .625 21 .857 17 .985 1 .00 10 .24 C ATOM 3626 CB ALA A 258 22..459 20,.568 17..233 1..00 9.96 c
ATOM 3630 C ALA A 258 22. .446 23, .027 17. .061 1. .00 11, .33 c
ATOM 3631 O ALA A 258 23. .426 23, .621 16. ,598 1, .00 10, .81 o
ATOM 3632 N GLY A 259 21. .191 23, .351 16. ,799 1. .00 11, .66 N
ATOM 3634 CA GLY A 259 20. .929 24 .467 15. .916 1, .00 12 .69 C
ATOM 3637 C GLY A 259 21. .378 25 .792 16, .529 1, .00 11, .32 C
ATOM 3638 O GLY A 259 21, .928 26, .653 15. .830 1. .00 12, .25 O
ATOM 3639 N LEU A 260 21. .044 25, .991 17. .781 1. .00 10, .90 N
ATOM 3641 CA LEU A 260 21, .453 27 .191 18, .479 1, .00 11 .40 C
ATOM 3643 CB LEU A 260 20, .945 27 .240 19, .877 1, .00 10 .96 C
ATOM 3646 CG LEU A 260 21, .372 28, .483 20. .678 1, .00 11. .84 C
ATOM 3648 CDl LEU A 260 20. .781 29, .683 20. .099 1, .00 16, .22 C
ATOM 3652 CD2 LEU A 260 20. .948 28, .349 22. ,174 1. .00 12, .84 C
ATOM 3656 C LEU A 260 22, .984 27 .291 18. .477 1, .00 12 .53 C
ATOM 3657 O LEU A 260 23 .558 28 .384 18, .205 1 .00 11 .28 O
ATOM 3658 N ALA A 261 23 .644 26 .153 18, .745 1 .00 11 .39 N
ATOM 3660 CA ALA A 261 25, .093 26 .133 18, .661 1. .00 11 .97 C
ATOM 3662 CB ALA A 261 25, .604 24 .716 18. .889 1, .00 13 .65 C
ATOM 3666 C ALA A 261 25 .607 26 .638 17, .292 1 .00 12 .95 C
ATOM 3667 O ALA A 261 26 .563 27 .413 17, .203 1, .00 12 .12 O
ATOM 3668 N ALA A 262 24, .998 26 .144 16, .229 1. .00 11 .02 N
ATOM 3670 CA ALA A 262 25, .435 26 .520 14, .869 1, .00 12 .24 C
ATOM 3672 CB ALA A 262 2 .731 25 .669 13, .828 1 .00 12 .25 C
ATOM 3676 C ALA A 262 25 .200 28 .002 14, .619 1 .00 13 .52 c
ATOM 3677 O ALA A 262 26, .045 28 .684 13, .996 1, .00 13 .55 o
ATOM 3678 N LYS A 263 24, .091 28 .505 15. .135 1, .00 13 .42 N
ATOM 3680 CA LYS A 263 23, .730 29, .913 15. .021 1, .00 13, .54 C
ATOM 3682 CB LYS A 263 22. .319 30 .164 15, .511 1 .00 14 .20 C
ATOM 3685 CG LYS A 263 21, .797 31 .603 15, .356 1, .00 13 .78 C
ATOM 3688 CD LYS A 263 20, .412 31, .725 15. .984 1, .00 15 .37 C
ATOM 3691 CE LYS A 263 19, .936 33, .158 16. .011 1, .00 17, .70 C
ATOM 3694 NZ LYS A 263 19 .671 33 .740 14, .618 1 .00 1 .92 N
ATOM 3698 C LYS A 263 24, .754 30 .774 15, .736 1, .00 12 .81 C
ATOM 3699 O LYS A 263 25, .225 31 .781 15. .148 1, .00 13 .25 O
ATOM 3700 N ILE A 264 25, .133 30, .393 16. .961 1, .00 12, .96 N
ATOM 3702 CA ILE A 264 26, .107 31, .136 17. .723 1, .00 12, .47 C
ATOM 3704 CB ILE A 264 26 .209 30 .579 19, .150 1, .00 12 .83 C
ATOM 3706 CGI ILE A 264 24, .895 30 .799 19. .927 1, .00 12 .96 C
ATOM 3709 CDl ILE A 264 24, .829 30, .017 21. .259 1, .00 13, .94 C
ATOM 3713 CG2 ILE A 264 27, .382 31, .146 19. .925 1, .00 12, .54 C
ATOM 3717 C ILE A 264 27, .478 31, .081 17. ,018 1. .00 12. .78 C
ATOM 3718 O ILE A 264 28, .147 32 .110 16. .835 1, .00 12 .72 o
ATOM 3719 N TRP A 265 27, .843 29. .890 16. .527 1, .00 12 .74 N
ATOM 3721 CA TRP A 265 29, .194 29, .711 15. .984 1, .00 13, .37 C
ATOM 3723 CB TRP A 265 29, .445 28, .252 15. .665 1, .00 13, .60 C
ATOM 3726 CG TRP A 265 30 .859 27 .836 15. ,758 1, .00 1 .37 C
ATOM 3727 CDl TRP A 265 31, .987 28 .624 15. ,964 1, .00 12 .89 C
ATOM 3729 NEl TRP A 265 33, .101 27, .823 16. ,063 1, .00 14, .49 N
ATOM 3731 CE2 TRP A 265 32, .705 26, .514 15. .998 1, .00 16, .16 C
ATOM 3732 CD2 TRP A 265 31, .313 26, .493 15. ,796 1. .00 12, .95 C
ATOM 3733 CE3 TRP A 265 30, .679 25 .254 15. .657 1, .00 14 .36 C
ATOM 3735 CZ3 TRP A 265 31, .472 24, .110 15. .707 1, .00 12, .68 C
ATOM 3737 CH2 TRP A 265 32, .809 24, .180 15. .941 1, ,00 12, .39 C
ATOM 3739 CZ2 TRP A 265 33, .462 25, .359 16. .069 1, ,00 15, .97 C
ATOM 3741 C TRP A 265 29 .378 30 .560 14. .732 1, .00 13, .71 C
ATOM 3742 O TRP A 265 30, .423 31, .117 14. .530 1, .00 17, .12 O
ATOM 3743 N SER A 266 28, .330 30, .599 13. .922 1, ,00 15, .25 N
ATOM 3745 CA SER A 266 28, .307 31, .345 12. ,676 1. ,00 14, .75 C
ATOM 3747 CB SER A 266 27, .000 31, .130 11. ,945 1. .00 15, .47 C
ATOM 3750 OG SER A 266 26 .987 31 .917 10. .741 1, .00 18, .19 O
ATOM 3752 C SER A 266 28 .498 32 .835 12, .962 1 .00 16 .77 C
ATOM 3753 O SER A 266 29 .218 33 .537 12, .227 1 .00 16 .07 O
ATOM 3754 N ALA A 267 27 .903 33 .307 14, .053 1. .00 15 .63 N ATOM 3756 CA ALA A 267 28..046 34..711 14..450 1..00 17..65 C
ATOM 3758 CB ALA A 267 26. .911 35. .093 15. .318 1. .00 19. .01 c
ATOM 3762 C ALA A 267 29. .405 35. .056 15. .092 1. 00 19. ,17 c
ATOM 3763 O ALA A 267 29. ,744 36, .249 15. 277 1. 00 18. .66 o
ATOM 3764 N ASN A 268 30. .188 34. .053 15. ,465 1. ,00 17. ,88 N
ATOM 3766 CA ASN A 268 31. .557 34. .318 15. ,886 1. ,00 20. ,05 C
ATOM 3768 CB ASN A 268 31. .688 34. .603 17. ,324 1. ,00 20. ,47 C
ATOM 3771 CG ASN A 268 33. .120 35. .029 17. ,701 1. ,00 23. ,58 C
ATOM 3772 OD1 ASN A 268 34. ,017 35, .076 16. .860 1. ,00 24. ,59 0
ATOM 3773 ND2 ASN A 268 33. .327 35, .286 18. .961 1. ,00 27. ,93 N
ATOM 3776 C ASN A 268 32. .437 33, .156 15. .520 1. ,00 18. ,98 C
ATOM 3777 O ASN A 268 32. .651 32, .264 16. .321 1. ,00 17. ,09 O
ATOM 3778 N THR A 269 32. .915 33 , .169 14. .289 1. ,00 19. ,21 N
ATOM 3780 CA THR A 269 33, .696 32 .076 13. .772 1. .00 19. .25 C
ATOM 3782 CB THR A 269 33, .838 32, .153 12. .207 1. .00 21. .27 C
ATOM 3784 OGl THR A 269 34, .379 33 .443 11. .889 1. .00 21, ,88 O
ATOM 3786 CG2 THR A 269 32, .479 32, .127 11. .565 1. .00 23, ,50 C
ATOM 3790 C THR A 269 35, ,055 32, .025 14. .380 1. ,00 19. ,11 C
ATOM 3791 O THR A 269 35 .761 31 .117 14, .072 1. .00 19, ,77 0
ATOM 3792 N SER A 270 35 .435 32 .929 15, .297 1. .00 19, ,17 N
ATOM 3794 CA SER A 270 36, .732 32 .774 15, .936 1. .00 19, ,54 C
ATOM 3796 CB . P-SER A 270 37 .231 34 .105 16, .454 0. .50 19, ,48 C
ATOM 3797 CB : BSER A 270 37 .264 34 .116 16 .400 0. .50 19 .66 C
ATOM 3802 OG . ASER A 270 36 .235 34 .749 17, .225 0, .50 21 .34 O
ATOM 3803 OG : BSER A 270 37 .628 34 .922 15, .280 0, .50 22 ,88 O
ATOM 3806 C SER A 270 36, .679 31, .795 17. .100 1. .00 17, .42 C
ATOM 3807 O SER A 270 37, .688 31, .382 17. .637 1. .00 19. .69 O
ATOM 3808 N LEU A 271 35 .493 31 .392 17, .484 1. .00 15. .84 N
ATOM 3810 CA LEU A 271 35 .364 30 .456 18, .595 1, .00 13, .46 C
ATOM 3812 CB LEU A 271 33, .910 30 .345 19, .026 1. .00 14, .65 C
ATOM 3815 CG LEU A 271 33, .146 31 .575 19, .466 1. .00 15, ,68 C
ATOM 3817 CDl LEU A 271 31 .659 31 .196 19. .782 1, .00 18 .69 C
ATOM 3821 CD2 LEU A 271 33 .807 32 .216 20, .685 1, .00 21 .20 C
ATOM 3825 C LEU A 271 35 .806 29 .028 18, .220 1. .00 12, .04 C
ATOM 3826 O LEU A 271 35 .573 28 .546 17, .091 1. .00 11, ,86 0
ATOM 3827 N SER A 272 36 .323 28 .335 19, .220 1. .00 12, ,57 N
ATOM 3829 CA SER A 272 36 .545 26 .919 19 .145 1. .00 11 .98 C
ATOM 3831 CB SER A 272 37 .710 26 .489 20 .040 1, .00 12 .49 C
ATOM 3834 OG SER A 272 37 .433 26 .778 21, .421 1, .00 12 .53 O
ATOM 3836 C SER A 272 35 .275 26 .244 19, .663 1, .00 11 .47 C
ATOM 3837 O SER A 272 34 .372 26 .889 20, .269 1, .00 9 .92 O
ATOM 3838 N HIS A 273 35 .225 24 .937 19 .454 1 .00 11 .48 N
ATOM 3840 CA HIS A 273 34 .074 24 .187 19 .920 1 .00 11 .75 C
ATOM 3842 CB HIS A 273 34 .187 22 .750 19 .445 1 .00 13 .59 C
ATOM 3845 CG HIS A 273 35 .160 21 .921 20 .197 1. .00 13 .01 C
ATOM 3846 NDl HIS A 273 36 .456 22 .312 20 .379 1. .00 11 .73 N
ATOM 3848 CEl HIS A 273 37 .072 21 .402 21 .124 1 .00 13 .52 C
ATOM 3850 NE HIS A 273 36 .243 20 .394 21 .343 1 .00 12 .58 N
ATOM 3852 CD2 HIS A 273 35 .039 20 .708 20 .787 1 .00 14 .38 C
ATOM 3854 C HIS A 273 33 .887 24 .268 21, .427 1, .00 11 .79 C
ATOM 3855 O HIS A 273 32 .723 24 .252 21 .930 1 .00 10 .83 O
ATOM 3856 N SER A 274 34 .975 24 .276 22 .191 1 .00 10 .50 N
ATOM 3858 CA SER A 274 34 .813 24 .319 23 .643 1 .00 10 .49 C
ATOM 3860 CB SER A 274 36 .035 23 .803 24 .397 1 .00 13 .18 C
ATOM 3863 OG SER A 274 37 .161 24 .639 24 .166 1 .00 12 .23 O
ATOM 3865 C SER A 274 34 .478 25 .702 24 .113 1 .00 12 .50 C
ATOM 3866 O SER A 274 33 .783 25 .849 25 .123 1 .00 11 .08 O
ATOM 3867 N GLN A 275 34 .919 26 .739 23 .418 1 .00 11 .42 N
ATOM 3869 CA GLN A 275 34 .425 28 .072 23 .784 1 .00 11 .21 C
ATOM 3871 CB GLN A 275 35 .132 29 .188 22 .999 1 .00 11 .23 C
ATOM 3874 CG GLN A 275 36 .608 29 .464 23 .375 1 .00 13 .15 C
ATOM 3877 CD GLN A 275 37 .192 30 .473 22 .413 1 .00 13 .10 C
ATOM 3878 OEl GLN A 275 37 .323 30 .182 21 .228 1 .00 15 .35 O ATOM 3879 NE2 GLN A 275 37,.555 31,.677 22.,923 1.,00 11..91 N
ATOM 3882 C GLN A 275 32. .898 28. .177 23. ,457 1. ,00 11. .10 C
ATOM 3883 O GLN A 275 32. .150 28. ,776 24. 217 1. 00 10. .23 O
ATOM 3884 N LEU A 276 32. .492 27. ,556 22. 340 1. 00 10. .08 N
ATOM 3886 CA LEU A 276 31, ,067 27. .521 21. ,932 1. ,00 10. .49 C
ATOM 3888 CB LEU A 276 30, ,909 26. .866 20. ,582 1. ,00 10. .65 C
ATOM 3891 CG LEU A 276 29, ,466 26. .606 20. 164 1. .00 8. .36 C
ATOM 3893 CDl LEU A 276 28. ,715 27. .856 20. 038 1. ,00 9. .01 C
ATOM 3897 CD2 LEU A 276 29. .489 25, .869 18, .849 1. ,00 11. .63 C
ATOM 3901 C LEU A 276 30. .272 26, .790 23, ,041 1, ,00 11. .06 C
ATOM 3902 O LEU A 276 29. .226 27, .252 23. ,494 1, ,00 10. .87 O
ATOM 3903 N ARG A 277 30. .783 25. .671 23. ,524 1. ,00 9, .13 N
ATOM 3905 CA ARG A 277 30. .104 24. .934 24. ,560 1. ,00 11. .95 C
ATOM 3907 CB ARG A 277 30. .915 23, .671 24. ,913 1. .00 10, .95 C
ATOM 3910 CG ARG A 277 30. .335 22 , .793 26. .018 1. .00 13, .22 C
ATOM 3913 CD ARG A 277 31. .103 21, .490 26. ,229 1. .00 13, .28 C
ATOM 3916 NE ARG A 277 30, .415 20, .601 27. ,149 1. .00 13, .25 N
ATOM 3918 CZ ARG A 277 30. .892 19, .416 27. ,518 1. ,00 14, .34 C
ATOM 3919 NHl ARG A 277 32, .089 19 .014 27. .110 1. .00 14 .38 N
ATOM 3922 NH2 ARG A 277 30, .190 18 .660 28. .345 1. .00 13 .28 N
ATOM 3925 C ARG A 277 29, .974 25, .722 25. .858 1. .00 12, .12 C
ATOM 3926 O ARG A 277 28, .949 25, .722 26. .538 1. .00 12, .16 O
ATOM 3927 N THR A 278 31. .023 26 .469 26, ,175 1, ,00 12 .13 N
ATOM 3929 CA THR A 278 31 .049 27 .295 27, .359 1. .00 11 .62 C
ATOM 3931 CB THR A 278 32 .461 27 .918 27, .485 1. .00 12 .51 C
ATOM 3933 OGl THR A 278 33. ,379 26. .892 27. .846 1. ,00 15, .78 O
ATOM 3935 CG2 THR A 278 32. ,551 28, .829 28. ,624 1. ,00 17, .34 C
ATOM 3939 C THR A 278 29, .990 28 .388 27. .287 1. .00 10 .34 C
ATOM 3940 O THR A 278 29, .372 28, .715 28. .294 1. .00 11, .22 O
ATOM 3941 N GLU A 279 29, .775 28, .943 26. .101 1. .00 10, .24 N
ATOM 3943 CA GLU A 279 28, .804 30, .034 25. .920 1. .00 10, .36 C
ATOM 3945 CB GLU A 279 29, .090 30, .744 24. .607 1. .00 13, .04 C
ATOM 3948 CG GLU A 279 28, .155 31 .799 24, .172 1, .00 13 .67 C
ATOM 3951 CD GLU A 279 27, .827 32 .923 25. .148 1, .00 11 .41 C
ATOM 3952 OEl GLU A 279 28, .413 33 .084 26. .245 1. .00 12 .14 O
ATOM 3953 OE2 GLU A 279 26, .928 33, .691 24. .766 1. .00 13, .94 O
ATOM 3954 C GLU A 279 27 .387 29 .439 25, .978 1, .00 10 .50 C
ATOM 3955 O GLU A 279 26 .477 30 .093 26, .462 1, .00 10 .16 O
ATOM 3956 N LEU A 280 27 .198 28 .205 25, ,499 1, .00 10 .73 N
ATOM 3958 CA LEU A 280 25, .898 27 .507 25, .652 1, .00 10 .31 C
ATOM 3960 CB LEU A 280 25, .846 26 .124 24, .973 1, .00 12 .52 C
ATOM 3963 CG LEU A 280 25 .772 26 .107 23, .451 1, .00 13 .85 C
ATOM 3965 CDl LEU A 280 25 .964 24 .631 22, .911 1, .00 17 .63 C
ATOM 3969 CD2 LEU A 280 24 .435 26 .669 22, .957 1, .00 15 .10 C
ATOM 3973 C LEU A 280 25 .613 27 .411 27, .150 1, .00 10 .89 C
ATOM 3974 O LEU A 280 24 .492 27 .626 27 .606 1 .00 12 .00 O
ATOM 3975 N GLN A 281 26 .630 27 .040 27 .907 1. .00 9 .57 N
ATOM 3977 CA GLN A 281 26 .479 26 .931 29 .322 1 .00 11 .52 C
ATOM 3979 CB GLN A 281 27 .755 26 .360 29 .925 1 .00 11 .03 C
ATOM 3982 CG GLN A 281 28, .028 24 .891 29. ,487 1. ,00 12 .22 C
ATOM 3985 CD GLN A 281 29 .376 24 .377 29, .981 1, .00 14 .98 C
ATOM 3986 OEl GLN A 281 30 .115 25 .172 30, .555 1, .00 15 .61 O
ATOM 3987 NE2 GLN A 281 29 .731 23 .103 29, .697 1, .00 9 .95 N
ATOM 3990 C GLN A 281 26 .161 28 .313 29, .924 1, .00 10 .71 C
ATOM 3991 O GLN A 281 25 .309 28 .409 30, .815 1, .00 11 .77 O
ATOM 3992 N ASN A 282 26 .883 29 .367 29 .484 1 .00 10 .78 N
ATOM 3994 CA ASN A 282 26 .722 30 .715 30, .090 1. .00 10 .24 C
ATOM 3996 CB ASN A 282 27 .683 31 .745 29, .504 1, .00 10 .08 C
ATOM 3999 CG ASN A 282 29 .136 31 .482 29, .812 1, .00 13 .02 C
ATOM 4000 OD1 ASN A 282 29 .471 30 .830 30 .809 1 .00 14 .49 O
ATOM 4001 ND2 ASN A 282 30 .003 32 .000 28 .973 1 .00 12 .29 N
ATOM 4004 C ASN A 282 25 .275 31 .172 29 .788 1 .00 11 .08 C
ATOM 4005 O ASN A 282 24 .588 31 .667 30 .681 1 .00 12 .10 O ATOM 4006 N ARG A 283 24.,829 30..902 28.,575 1..00 9..30 N
ATOM 4008 CA ARG A 283 23. ,484 31. .298 28. ,160 1. .00 10. .89 C
ATOM 4010 CB ARG A 283 23. ,305 31. .055 26. ,697 1. .00 10. .96 C
ATOM 4013 CG ARG A 283 23, ,894 32. ,191 25. ,842 1. ,00 13. .15 C
ATOM 4016 CD ARG A 283 23. .768 31. .880 24. .383 1, .00 11, .91 C
ATOM 4019 NE ARG A 283 24. .469 32. .831 23. .466 1, .00 12, .89 N
ATOM 4021 CZ ARG A 283 23. .985 33. .290 22. .311 1, .00 15, .41 C
ATOM 4022 NHl ARG A 283 22. .780 33. .006 21. .903 1. .00 15, .22 N
ATOM 4025 NH2 ARG A 283 24. .722 34. .053 21. .528 1. .00 14, .46 N
ATOM 4028 C ARG A 283 22. .450 30. .528 28. .963 1. .00 10, .28 C
ATOM 4029 O ARG A 283 21. .390 31. .063 29. .298 1. .00 11, ,49 O
ATOM 4030 N ALA A 284 22. .741 29. .263 29. .220 1. .00 10, ,27 N
ATOM 4032 CA ALA A 284 21. .789 28. .438 29. .957 1. .00 10, .07 C
ATOM 4034 CB ALA A 284 22, .358 27, .011 30. .158 1, .00 11 ,06 C
ATOM 4038 C ALA A 284 21, .493 29, .093 31, .306 1, .00 10 .53 C
ATOM 4039 O ALA A 284 20, .349 29, .096 31, .809 1, .00 11. ,23 O
ATOM 4040 N LYS A 285 22, .539 29, .620 31, .899 1, .00 10, ,42 N
ATOM 4042 CA LYS A 285 22, .446 30, .158 33. .238 1, .00 10, ,99 C
ATOM 4044 CB LYS A 285 23 .846 30, .372 33, .821 1 .00 11 .62 C
ATOM 4047 CG LYS A 285 24 .664 29, .120 34, .054 1. .00 12 .18 c
ATOM 4050 CD LYS A 285 26, .057 29. ,529 34. .477 1. .00 16, .21 c
ATOM 4053 CE LYS A 285 27. .062 28. .492 34. .451 1. .00 15, ,01 c
ATOM 4056 NZ LYS A 285 28. .349 29, .067 35, .077 1, .00 17 .35 N
ATOM 4060 C LYS A 285 21, .566 31, .386 33, .281 1, .00 11 .59 C
ATOM 4061 O LYS A 285 21, .120 31, .753 34, .363 1, .00 12 .70 O
ATOM 4062 N VAL A 286 21, .419 32, .094 32, .168 1, .00 10, .88 N
ATOM 4064 CA VAL A 286 20, .604 33, .255 32, .110 1, .00 12, .69 C
ATOM 4066 CB VAL A 286 20 .719 33 .961 30, .788 1 .00 12 .21 C
ATOM 4068 CGI VAL A 286 19 .838 35, .220 30, .746 1 .00 15 .12 C
ATOM 4072 CG2 VAL A 286 22, .221 34, .402 30, .612 1 .00 13 .05 C
ATOM 4076 C VAL A 286 19, .153 32, .850 32, .362 1. .00 13 .03 C
ATOM 4077 O VAL A 286 18, .399 33, .651 32, .960 1, .00 13 .04 o
ATOM 4078 N TYR A 287 18, .788 31, .649 31, .885 1, .00 11, .44 N
ATOM 4080 CA TYR A 287 17, .427 31, .124 32, .057 1, .00 12, .02 C
ATOM 4082 CB TYR A 287 16, .740 30, .949 30, .718 1, .00 12, .84 C
ATOM 4085 CG TYR A 287 16, ,587 32, .235 29, .976 1, .00 12, .74 C
ATOM 4086 CDl TYR A 287 15 .615 33 .157 30 .344 1. .00 14 .60 c
ATOM 4088 CEl TYR A 287 15 .490 34 .343 29 .748 1 .00 14 .77 c
ATOM 4090 CZ TYR A 287 16 .343 34. .709 28 .753 1 .00 15 .62 c
ATOM 4091 OH TYR A 287 16 .139 35 .938 28. .183 1 .00 18 .73 0
ATOM 4093 CE2 TYR A 287 17 .372 33 .852 28, .377 1 .00 13 .12 c
ATOM 4095 CD2 TYR A 287 17 .521 32 .646 28 .995 1 .00 11 .74 c
ATOM 4097 C TYR A 287 17 .427 29 .860 32 .905 1 .00 12 .13 c
ATOM 4098 O TYR A 287 17 .303 28, .740 32, .452 1 .00 13 .99 0
ATOM 4099 N ASP A 288 17, .631 30, .057 34, .208 1, .00 12 .75 N
ATOM 4101 CA ASP A 288 17 .553 29 .009 35 .170 1 .00 11 .58 C
ATOM 4103 CB ASP A 288 17 .721 29 .636 36 .551 1 .00 11 .65 C
ATOM 4106 CG ASP A 288 17 .912 28 .612 37 .687 1 .00 15 .60 c
ATOM 4107 OD1 ASP A 288 18 .065 27 .373 37 .473 1 .00 17 .35 0
ATOM 4108 OD2 ASP A 288 17 .934 29 .001 38 .898 1 .00 15 .75 0
ATOM 4109 C ASP A 288 16 .174 28 .337 35 .056 1 .00 12 .45 c
ATOM 4110 O ASP A 288 15 .186 29 .032 34 .950 1 .00 13 .43 o
ATOM 4111 N ILE A 289 16 .112 27 .026 34 .986 1 .00 10 .72 N
ATOM 4113 CA ILE A 289 14 .812 26 .271 34 .969 1 .00 12 .80 C
ATOM 4115 CB ILE A 289 14 .860 25 .168 33 .912 1 .00 12 .78 C
ATOM 4117 CGI ILE A 289 15 .042 25 .785 32 .535 1 .00 13 .67 C
ATOM 4120 CDl ILE A 289 13 .745 26 .474 31 .867 1 .00 13 .36 c
ATOM 4124 CG2 ILE A 289 13 .659 24 .220 33 .989 1 .00 14 .37 c
ATOM 4128 C ILE A 289 14 .517 25 .742 36 .352 1 .00 13 .79 c
ATOM 4129 O ILE A 289 15 .376 25 .140 37 .045 1 .00 12 .95 0
ATOM 4130 N LYS A 290 13 .299 26 .006 36 .825 1 .00 14 .91 N
ATOM 4132 CA LYS A 290 12 .904 25 .675 38 .185 1 .00 16 .59 C
ATOM 4134 CB LYS A 290 12 .600 26 .955 38 .930 1 .00 17 .67 C ATOM 4137 CG LYS A 290 13.878 27.786 39.143 1.00 21.86 c
ATOM 4140 CD LYS A 290 13 .627 28 .893 40 .134 1 .00 28 .07 c
ATOM 4143 CE LYS A 290 13, .554 30 .215 39, .470 1 .00 34 .10 c
ATOM 4146 NZ LYS A 290 13, .272 31 .317 40, .512 1. .00 39 .60 N
ATOM 4150 C LYS A 290 11 .648 24 .807 38 .235 1 .00 16 .71 C
ATOM 4151 O LYS A 290 11 .200 24 .444 39 .330 1 .00 18 .64 O
ATOM 4152 N GLY A 291 11, .119 24 .507 37, .071 1 .00 15 .39 N
ATOM 4154 CA GLY A 291 9, .895 23, .740 36, .907 1, .00 14 .24 C
ATOM 4157 C GLY A 291 10 .134 22 .294 36, .570 1 .00 14 .85 C
ATOM 4158 O GLY A 291 10 .997 21 .990 35. .707 1 .00 15 .05 O
ATOM 4159 N GLY A 292 9, .376 21. .387 37, .185 1 .00 15 .54 N
ATOM 4161 CA GLY A 292 9, .524 19, .984 36, .896 1, .00 14, .12 C
ATOM 4164 C GLY A 292 10, .116 19, .187 38, .030 1, .00 15, .07 C
ATOM 4165 O GLY A 292 10, .717 19 .734 38, .959 1, .00 15 .42 O
ATOM 4166 N ILE A 293 9 .932 17 .878 37 .980 1 .00 14 .74 N
ATOM 4168 CA ILE A 293 10 .363 17 .018 39 .044 1 .00 15 .41 C
ATOM 4170 CB ILE A 293 9, .881 15 .599 38, .776 1 .00 15 .32 C
ATOM 4172 CGI ILE A 293 8, .342 15, .549 38, .894 1, .00 18 .82 C
ATOM 4175 CDl ILE A 293 7 .731 14 .260 38 .471 1 .00 21 .20 C
ATOM 4179 CG2 ILE A 293 10 .526 14 .610 39 .740 1 .00 15 .00 C
ATOM 4183 C ILE A 293 11, .885 17 .052 39, .176 1 .00 16 .26 C
ATOM 4184 O ILE A 293 12, .586 16, .688 38, .214 1, .00 14 .62 O
ATOM 4185 N GLY A 294 12 .367 17 .439 40 .346 1 .00 14 .72 N
ATOM 4187 CA GLY A 294 13, .810 17 .581 40, .559 1 .00 15 .36 C
ATOM 4190 C GLY A 294 14, .449 18, .897 40, .107 1, .00 13 .63 C
ATOM 4191 O GLY A 294 15, .660 19, .095 40, .315 1, .00 15, .06 O
ATOM 4192 N ALA A 295 13. .688 19, .803 39. .516 1, .00 14, .00 N
ATOM 4194 CA ALA A 295 14, .220 21, .064 39. .058 1, .00 13, .92 C
ATOM 4196 CB ALA A 295 13, .381 21, .661 37. .869 1, .00 14, .66 C
ATOM 4200 C ALA A 295 14, .226 21 .990 40, .236 1 .00 16 .73 C
ATOM 4201 O ALA A 295 13, .451 21, .811 41, .178 1, .00 16 .12 O
ATOM 4202 N GLY A 296 15, .094 22, .986 40, .227 1 .00 16 .79 N
ATOM 4204 CA GLY A 296 15, .114 23 .941 41, .340 1 .00 18 .72 C
ATOM 4207 C GLY A 296 16, .052 25, .058 41, .052 1, .00 17, .66 C
ATOM 4208 O GLY A 296 16, .601 25, .160 39. .963 1, .00 16, .21 O
ATOM 4209 N THR A 297 16. .240 25. .979 41. .992 1, .00 17, .88 N
ATOM 4211 CA THR A 297 17, .144 27, .068 41. .712 1, .00 17 .28 C
ATOM 4213 CB THR A 297 17. .117 27, .981 42. .941 1, .00 19, .91 C
ATOM 4215 OGl THR A 297 15. .763 28, .455 43. .102 1, .00 19, .89 0
ATOM 4217 CG2 THR A 297 17. .981 29. .177 42. .689 1. .00 19, .92 c
ATOM 4221 C THR A 297 18. .563 26. .600 41. .472 1. .00 17. .13 c
ATOM 4222 O THR A 297 19. .104 25. .832 42, .248 1. .00 18, .37 o
ATOM 4223 N GLY A 298 19. .193 27. .074 40, .410 1, .00 14, .91 N
ATOM 4225 CA GLY A 298 20. .569 26. .702 40. .153 1. .00 15, .10 C
ATOM 4228 C GLY A 298 20. .684 25. .492 39. .273 1. .00 14, ,01 C
ATOM 4229 O GLY A 298 19 .717 25 .039 38 .656 1 .00 12 .62 O
ATOM 4230 N ASP A 299 21 .882 24 .932 39 .243 1 .00 13 .64 N
ATOM 4232 CA ASP A 299 22. .189 23 .741 38 .460 1 .00 13 .68 C
ATOM 4234 CB ASP A 299 23, .689 23, .623 38, .447 1, .00 14, .28 C
ATOM 4237 CG ASP A 299 24, .229 22, .342 37, .843 1. .00 15, .32 C
ATOM 4238 OD1 ASP A 299 23 .578 21 .787 36 .970 1 .00 13 .87 O
ATOM 4239 OD2 ASP A 299 25, .330 21, .885 38. .238 1 .00 13 .70 O
ATOM 4240 C ASP A 299 21, .507 22 , .602 39, .092 1, .00 14, .87 C
ATOM 4241 O ASP A 299 21, .657 22, .381 40, .306 1, .00 16, .32 O
ATOM 4242 N ASP A 300 20. .664 21, .901 38, .348 1, .00 12, .69 N
ATOM 4244 CA ASP A 300 19. .921 20, .812 38, .953 1, .00 12, .30 C
ATOM 4246 CB ASP A 300 18. .489 21, .282 39, .369 1, .00 12, .84 C
ATOM 4249 CG ASP A 300 17, .615 21, .597 38, .192 1, .00 11, .72 C
ATOM 4250 OD1 ASP A 300 17. .330 20. .687 37, .428 1, .00 13, .65 O
ATOM 4251 OD2 ASP A 300 17, .079 22, .691 38, .018 1 .00 14, .33 O
ATOM 4252 C ASP A 300 19, .928 19, .603 38 .011 1. .00 13 .91 C
ATOM 4253 O ASP A 300 20, .278 19 .699 36 .822 1 .00 13 .63 o
ATOM 4254 N TYR A 301 19, .514 18 .462 38, .539 1 .00 13 .49 N ATOM 4256 CA TYR A 301 19.627 17.220 37.803 .00 12.93 C
ATOM 4258 CB TYR A 301 19.868 16.046 38.777 .00 11.22 C
ATOM 4261 CG TYR A 301 18.804 15.846 39.823 .00 13.88 C
ATOM 4262 CDl TYR A 301 17.648 15.225 39.523 .00 14.91 C
ATOM 4264 CEl TYR A 301 16.621 15.079 40.531 .00 17.24 C
ATOM 4266 CZ TYR A 301 16.809 15.580 41.788 .00 19.26 C
ATOM 4267 OH TYR A 301 15.803 15.394 42.776 .00 20.79 O
ATOM 4269 CE TYR A 301 17.977 16.242 42.081 .00 16.95 C
ATOM 4271 CD2 TYR A 301 .948 16.384 41.095 .00 13.89 C
ATOM 4273 C TYR A 301 .421 16.951 36.875 .00 11.64 C
ATOM 4274 O TYR A 301 .419 15.954 36.154 1.00 11.11 O
ATOM 4275 N ALA A 302 17.435 17.846 36.832 00 11.29 N
ATOM 4277 CA ALA A 302 16.389 17.756 35.829 00 10.84 C
ATOM 4279 CB ALA A 302 15.006 18.227 36.391 00 12.99 C
ATOM 4283 C ALA A 302 16.684 18.574 34.581 00 10.79 C
ATOM 4284 O ALA A 302 16.424 18.146 33.444 00 11.72 O
ATOM 4285 N SER A 303 17.194 19.774 34.790 1.00 10.48 N
ATOM 4287 CA SER A 303 17.364 20.706 33.697 00 9.41 C
ATOM 4289 CB SER A 303 16.512 21.934 33.969 00 9.70 C
ATOM 4292 OG SER A 303 16.992 22.660 35.130 00 12 .05 O
ATOM 4294 C SER A 303 18.819 21.172 33.468 00 10. .16 C
ATOM 4295 O SER A 303 19.049 21.949 32.566 00 10, .53 O
ATOM 4296 N GLY A 304 19.742 20.739 34.303 00 8 .73 N
ATOM 4298 CA GLY A 304 21.132 21.139 34.201 00 10 .18 C
ATOM 4301 C GLY A 304 21.273 22.616 34.479 00 10 .17 C
ATOM 4302 O GLY A 304 20.710 23.138 35.422 00 10 .02 O
ATOM 4303 N PHE A 305 22.102 23.286 33.664 1.00 11.73 N
ATOM 4305 CA PHE A 305 22.452 24.644 33.880 1.00 10.68 C
ATOM 4307 CB PHE A 305 23.661 25.048 33.036 00 10.84 C
ATOM 4310 CG PHE A 305 24.977 24.418 33.419 00 11.70 C
ATOM 4311 CDl PHE A 305 25.070 23.280 34.195 00 12.20 C
ATOM 4313 CEl PHE A 305 26.316 22.689 34.511 00 12.74 C
ATOM 4315 CZ PHE A 305 27.499 23.265 33.973 00 11.93 C
ATOM 4317 CE2 PHE A 305 27.380 24.372 33.184 00 11.62 C
ATOM 4319 CD2 PHE A 305 26.157 24.941 32.891 00 12.26 C
ATOM 4321 C PHE A 305 21.349 25.614 33.554 00 11.33 C
ATOM 4322 O PHE A 305 21.368 26.748 34.022 00 11.39 O
ATOM 4323 N GLY A 306 20.364 25.161 32.811 00 10.40 N
ATOM 4325 CA GLY A 306 19.276 26.018 32.414 00 10.88 C
ATOM 4328 C GLY A 306 18.926 25.834 30.963 00 11.20 C
ATOM 4329 O GLY A 306 19.208 24.812 30.318 00 9.40 O
ATOM 4330 N TYR A 307 18.291 26.863 30.443 00 10.29 N
ATOM 4332 CA TYR A 307 17.803 26.905 29.077 00 11.10 C
ATOM 4334 CB TYR A 307 16.311 27.194 29.162 00 10.85 C
ATOM 4337 CG TYR A 307 15.557 27.309 27.870 00 10.79 C
ATOM 4338 CDl TYR A 307 16.087 26.885 26.660 00 10.35 C
ATOM 4340 CEl TYR A 307 15.374 27.001 25.479 1.00 10.97 C
ATOM 4342 CZ TYR A 307 14.086 27.458 25.503 1.00 13.85 C
ATOM 4343 OH TYR A 307 13.388 27.607 24.308 .00 13.18 O
ATOM 4345 CE2 TYR A 307 13.531 27.912 26.705 .00 13.38 C
ATOM 4347 CD2 TYR A 307 14.260 27.849 27.860 .00 11.68 C
ATOM 4349 C TYR A 307 18.479 27.939 28.217 ,00 12.67 C
ATOM 4350 O TYR A 307 18.140 29.113 28.271 .00 13.67 o
ATOM 4351 N PRO A 308 19.440 27.555 27.389 .00 11.31 N
ATOM 4352 CA PRO A 308 20.118 28.547 26.531 .00 11.31 C
ATOM 4354 CB PRO A 308 21.315 27.780 25.971 .00 10.82 C
ATOM 4357 CG PRO A 308 20.941 26.293 26.070 ,00 11.80 C
ATOM 4360 CD PRO A 308 19.833 26.179 27.077 1.00 12.92 C
ATOM 4363 C PRO A 308 19.245 29.003 25.361 00 11.34 C
ATOM 4364 O PRO A 308 18.604 28.159 24.689 00 10.74 0
ATOM 4365 N ARG A 309 19.314 30.266 25.051 00 12.86 N
ATOM 4367 CA ARG A 309 18.427 30.864 24.076 00 13.91 C
ATOM 4369 CB ARG A 309 17.252 31.527 24.844 00 13.02 C ATOM 4372 CG ARG A 309 16,.469 30,.638 25.,781 1.,00 13,.54 C
ATOM 4375 CD ARG A 309 15, .375 31 .251 26. .661 1. .00 14 .50 C
ATOM 4378 NE ARG A 309 14, .099 31 .449 25. ,978 1. ,00 15, .52 N
ATOM 4380 CZ ARG A 309 13. .022 31, .993 26. ,525 1. ,00 16, .18 C
ATOM 4381 NHl ARG A 309 13, .055 32 .423 27. .777 1. .00 12 .53 N
ATOM 4384 NH2 ARG A 309 11 .891 32 .006 25. .832 1, .00 15 .18 N
ATOM 4387 C ARG A 309 19, .139 31 .870 23. .213 1. .00 14 .57 C
ATOM 4388 O ARG A 309 20, .288 32 .256 23. .468 1. .00 13, .36 O
ATOM 4389 N VAL A 310 18 .455 32 .367 22, .180 1. .00 13 .70 N
ATOM 4391 CA VAL A 310 19 .053 33 .390 21, .325 1, .00 15 .05 C
ATOM 4393 CB VAL A 310 18 .134 33 .596 20, .101 1. .00 14 .71 C
ATOM 4395 CGI VAL A 310 18, .580 34 .793 19. .258 1. .00 18, .54 C
ATOM 4399 CG2 VAL A 310 18, .121 32, .368 19. ,265 1. ,00 14, .81 C
ATOM 4403 C VAL A 310 19, .272 34 .671 22, .112 1. .00 16 .45 C
ATOM 4404 O VAL A 310 20 .251 35 .382 21, .905 1. .00 15 .49 O
ATOM 4405 N LYS A 311 18 .316 34 .979 22, .979 1. .00 17 .77 N
ATOM 4407 CA LYS A 311 18, .415 36 .106 23. .940 1. .00 18, .52 C
ATOM 4409 CB LYS A 311 17, .942 37, .380 23. .244 1. ,00 18. .11 C
ATOM 4412 CG LYS A 311 16 .677 37 .137 22 .440 1, .00 23 .20 C
ATOM 4415 CD LYS A 311 15 .574 38 .112 22, .630 1. .00 33 .02 C
ATOM 4418 CE LYS A 311 15, .667 39 .252 21, .732 1. .00 34 .63 C
ATOM 4421 NZ LYS A 311 14, .289 39 .716 21. .277 1. .00 36, .21 N
ATOM 4425 C LYS A 311 17 .532 35 .897 25 .178 1, .00 17 .62 C
ATOM 4426 O LYS A 311 16 .770 34 .912 25, .199 1, .00 16 .50 O
ATOM 4427 OXT LYS A 311 17 .534 36 .644 26, .172 1, .00 19 .57 O
ATOM 4428 CA CA C 312 28, .232 18 .547 38. .069 1. .00 13, .89 CA
ATOM 4429 CA CA C 313 17 .608 25 .091 37, .856 1. .00 18 .60 CA
ATOM 4430 CA CA C 314 27 .338 14 .925 29, .013 0, .60 10 .25 CA
ATOM 4431 N ALA B 318 2. .727 2. .475 36, .156 1. .00 30 .60 N
ATOM 4433 CA ALA B 318 2, .319 3. .152 34. .902 1. .00 28, .10 C
ATOM 4435 CB ALA B 318 1, .428 4, .295 35. .200 1. .00 27, .75 C
ATOM 4439 C ALA B 318 3 .422 3 .533 33, .900 1. .00 24 .85 C
ATOM 4440 O ALA B 318 3, .103 3 .596 32, .739 1. .00 27 .46 O
ATOM 4443 N THR B 319 4, .625 3 .965 34. .273 1. .00 24. .93 N
ATOM 4445 CA THR B 319 5. ,658 4, .224 33. ,231 1. .00 23, .26 C
ATOM 4447 CB THR B 319 6, .154 5, .690 33. .296 1. .00 24, .03 C
ATOM 4449 OGl THR B 319 6, .811 5 .953 34, .535 1. .00 27 .37 O
ATOM 4451 CG2 THR B 319 4, .960 6. .670 33. .258 1. .00 27, .35 C
ATOM 4455 C THR B 319 6, ,926 3, .305 33. .235 1. .00 22. .75 C
ATOM 4456 O THR B 319 7, .820 3 .498 32, .406 1. .00 21 .26 O
ATOM 4457 N GLU B 320 7, .027 2 .401 34. .205 1. .00 21, .07 N
ATOM 4459 CA GLU B 320 8 .177 1 .559 34. .324 1, .00 21 .49 C
ATOM 4461 CB GLU B 320 9, .328 2 .280 35, .014 1. .00 21 .67 C
ATOM 4464 CG GLU B 320 8 .980 2 .681 36, .413 1, .00 25 .74 C
ATOM 4467 CD GLU B 320 10 .174 3 .130 37, .222 1, .00 34 .05 C
ATOM 4468 OEl GLU B 320 10 .962 3 .910 36, .698 1, .00 36 .87 O
ATOM 4469 OE2 GLU B 320 10 .295 2 .711 38, .394 1, .00 39 .11 O
ATOM 4470 C GLU B 320 7, .818 0 .308 35, .082 1, .00 20 .20 C
ATOM 4471 O GLU B 320 6 .914 0 .330 35, .945 1, .00 20 .85 O
ATOM 4472 N TRP B 321 8, .526 -0 .774 34, .748 1, .00 18 .06 N
ATOM 4474 CA TRP B 321 8 .271 -2 .092 35, .310 1, .00 17 .54 C
ATOM 4476 CB TRP B 321 7 .595 -2 .961 34, .277 1, .00 15 .93 C
ATOM 4479 CG TRP B 321 6 .265 -2 .537 33, .906 1, .00 18 .41 C
ATOM 4480 CDl TRP B 321 5 .089 -3 .007 34, .445 1, .00 16 .53 C
ATOM 4482 NEl TRP B 321 4. .017 -2 .406 33. .836 1 .00 19 .73 N
ATOM 4484 CE2 TRP B 321 4 .470 -1 .523 32, .886 1, .00 15 .64 C
ATOM 4485 CD2 TRP B 321 5 .889 -1 .576 32 .903 1 .00 15 .37 C
ATOM 4486 CE3 TRP B 321 6 .596 -0 .772 31, .992 1, .00 14 .76 C
ATOM 4488 CZ3 TRP B 321 5, .852 0 .097 31, .140 1, .00 17 .06 C
ATOM 4490 CH2 TRP B 321 4 .428 0 .089 31, .165 1, .00 17 .68 C
ATOM 4492 CZ2 TRP B 321 3 .757 -0 .693 32 .047 1 .00 18 .99 C
ATOM 4494 C TRP B 321 9 .570 -2 .716 35, .728 1 .00 16 .82 C
ATOM 4495 O TRP B 321 10 .068 -3 .654 35, .131 1, .00 16 .09 O ATOM 4496 N PRO B 322 10.186 -2.151 36..755 1,.00 18..33 N
ATOM 4497 CA PRO B 322 11 .493 -2 .628 37, .202 1, ,00 19. .01 C
ATOM 4499 CB PRO B 322 11, .829 -1, .725 38. ,392 1. ,00 19. .99 C
ATOM 4502 CG PRO B 322 10, .556 -1, .019 38. ,744 1. .00 19, .55 C
ATOM 4505 CD PRO B 322 9 .651 -1 .061 37, ,575 1. .00 19, .07 C
ATOM 4508 C PRO B 322 11 .484 -4 .065 37, .641 1, .00 19, .86 C
ATOM 4509 O PRO B 322 12, .495 -4, .748 37. ,546 1. ,00 20, .25 O
ATOM 4510 N GLU B 323 10, .334 -4, .557 38. .050 1. .00 20, ,04 N
ATOM 4512 CA GLU B 323 10 .262 -5 .906 38. ,546 1, .00 20 .93 C
ATOM 4514 CB GLU B 323 8, .960 -6 .041 39. ,331 1, .00 23, .24 C
ATOM 4517 CG GLU B 323 7, .708 -5, .954 38. .441 1. .00 26, .81 C
ATOM 4520 CD GLU B 323 7, .184 -4, .530 38. .089 1. .00 27, .51 C
ATOM 4521 OEl GLU B 323 7, .879 -3, .511 38. .275 1. .00 20. .70 o
ATOM 4522 OE2 GLU B 323 5, .996 -4, .461 37. .641 1. .00 30. .50 0
ATOM 4523 C GLU B 323 10 .325 -6 .934 37. .407 1, .00 18 .55 C
ATOM 4524 O GLU B 323 10 .521 -8 .111 37, .642 1, .00 16, .50 o
ATOM 4525 N LEU B 324 10, .256 -6 .485 36. ,172 1. .00 16, .08 N
ATOM 4527 CA LEU B 324 10, .357 -7, .398 35, ,050 1. .00 15, .61 C
ATOM 4529 CB LEU B 324 9 .626 -6 .849 33. ,846 1. .00 15 .54 C
ATOM 4532 CG LEU B 324 8 .113 -6 .779 34, ,039 1, ,00 16, .64 C
ATOM 4534 CDl LEU B 324 7, .437 -6. .061 32. ,923 1. ,00 19, .42 C
ATOM 4538 CD2 LEU B 324 7, .648 -8, .212 34. .152 1. .00 19, .22 C
ATOM 4542 C LEU B 324 11 .815 -7 .755 34. .643 1. ,00 14, .32 c
ATOM 4543 O LEU B 324 12 .017 -8 .619 33, .836 1. .00 14, .01 0
ATOM 4544 N VAL B 325 12, .792 -7, .073 35. ,189 1. .00 15, .66 N
ATOM 4546 CA VAL B 325 14, .180 -7, .370 34. .816 1. .00 15, ,56 C
ATOM 4548 CB VAL B 325 15, ,184 -6, .410 35. ,456 1. ,00 15. .71 C
ATOM 4550 CGI VAL B 325 16, ,629 -6 .860 35. .094 1. .00 16, .62 C
ATOM 4554 CG2 VAL B 325 14, .953 -4, .946 34. .988 1. .00 14, .67 C
ATOM 4558 C VAL B 325 14 .478 -8 .817 35, .197 1, .00 15 .70 C
ATOM 4559 O VAL B 325 14, .181 -9 .219 36. .316 1. ,00 15, .01 O
ATOM 4560 N GLY B 326 14, .985 -9 .609 34, .247 1, .00 16 .26 N
ATOM 4562 CA GLY B 326 15 .302 -11 .008 34, .494 1, .00 15 .67 C
ATOM 4565 C GLY B 326 14, .166 -11 .958 34. ,140 1. .00 16, .74 C
ATOM 4566 O GLY B 326 14, .358 -13, .159 34, ,108 1. .00 16, .04 O
ATOM 4567 N LYS B 327 12, .957 -11, .432 33. .950 1. .00 16, .30 N
ATOM 4569 CA LYS B 327 11, .848 -12 .266 33. .510 1, .00 17, .25 C
ATOM 4571 CB LYS B 327 10, .514 -11, .632 33. .958 1. .00 17, .70 C
ATOM 4574 CG LYS B 327 10, .573 -11, .307 35. .417 1. .00 22, .70 C
ATOM 4577 CD LYS B 327 9, .217 -11, .378 36. .140 1. .00 30, ,58 C
ATOM 4580 CE LYS B 327 9. .404 -11, .302 37. ,674 1. .00 31. ,61 C
ATOM 4583 NZ LYS B 327 10, .674 -11, .962 38. .027 1. .00 33, ,63 N
ATOM 4587 C LYS B 327 11, .837 -12, .497 32. .021 1. .00 16, .69 C
ATOM 4588 O LYS B 327 12, .479 -11, .805 31. ,222 1. .00 15, .76 O
ATOM 4589 N SER B 328 11. .055 -13, .485 31. ,601 1. ,00 18, .24 N
ATOM 4591 CA SER B 328 10 .972 -13 .809 30, ,188 1. .00 17 .59 C
ATOM 4593 CB SER B 328 10 .280 -15 .154 29, .994 1, .00 19 ,45 C
ATOM 4596 OG SER B 328 8 .887 -15 .026 30, .192 1, .00 18. ,39 O
ATOM 4598 C SER B 328 10 .206 -12 .757 29. .431 1, .00 18, ,63 C
ATOM 4599 O SER B 328 9, .375 -12 .020 30. .003 1, .00 17, ,45 O
ATOM 4600 N VAL B 329 10 .494 -12 .653 28. ,140 1, .00 18 ,81 N
ATOM 4602 CA VAL B 329 9 .771 -11 .690 27. ,299 1, .00 20, ,52 C
ATOM 4604 CB VAL B 329 10, .317 -11 .663 25, ,852 1, .00 21, .15 C
ATOM 4606 CGI VAL B 329 9, .817 -12, .809 25. .060 1. .00 22, .84 C
ATOM 4610 CG2 VAL B 329 9, .914 -10, .411 25. .159 1. .00 25, .35 C
ATOM 4614 C VAL B 329 8, .276 -11 .962 27. .279 1. .00 20, ,54 C
ATOM 4615 O VAL B 329 7, .460 -11 .049 27. .237 1, ,00 19, ,76 O
ATOM 4616 N GLU B 330 7, .906 -13, .228 27. .332 1, ,00 21, ,77 N
ATOM 4618 CA GLU B 330 6, .504 -13, .544 27. ,379 1, ,00 22, ,29 C
ATOM 4620 CB GLU B 330 6 .331 -15 .048 27. .153 1, .00 24, ,60 C
ATOM 4623 CG GLU B 330 6 .850 -15 .472 25, .754 1 .00 27 .68 C
ATOM 4626 CD GLU B 330 8 .341 -15 .854 25, .674 1. .00 35 .11 C
ATOM 4627 OEl GLU B 330 9 .141 -15 .647 26, .617 1, .00 28 .88 O ATOM 4628 OE2 GLU B 330 8..722 -16,.426 24,.632 1..00 41.94 0
ATOM 4629 C GLU B 330 5, .809 -13, .084 28, .642 1. .00 22 .11 C
ATOM 4630 O GLU B 330 4, .676 -12, .586 28. .584 1. .00 21. .27 o
ATOM 4631 N GLU B 331 6, .452 -13, .239 29. .790 1. .00 21, .03 N
ATOM 4633 CA GLU B 331 5 .904 -12, .751 31. .043 1, .00 21 .48 C
ATOM 4635 CB GLU B 331 6 .730 -13, .205 32. .262 1, .00 22 .76 C
ATOM 4638 CG GLU B 331 5, .844 -13, .545 33. .454 1. .00 31, .85 c
ATOM 4641 CD GLU B 331 6, .490 -13, .408 34. .816 1. .00 38, .17 c
ATOM 4642 OEl GLU B 331 7 .584 -14 .024 35 .014 1 .00 46 .35 o
ATOM 4643 OE2 GLU B 331 5 .886 -12 .712 35 .693 1, .00 37 .37 0
ATOM 4644 C GLU B 331 5 .838 -11 .243 31, .046 1, .00 19 .51 c
ATOM 4645 O GLU B 331 4 .892 -10 .668 31, .537 1, .00 18 .89 o
ATOM 4646 N ALA B 332 6 .882 -10, .609 30, .514 1, .00 18 .18 N
ATOM 4648 CA ALA B 332 6 .951 -9 .187 30, .538 1, .00 15 .95 C
ATOM 4650 CB ALA B 332 8 .254 -8 .709 29, .931 1, .00 16 .83 C
ATOM 4654 C ALA B 332 5 .801 -8 .640 29, .757 1, .00 15 .42 C
ATOM 4655 O ALA B 332 5, .163 -7, .697 30, .182 1. .00 14 .99 O
ATOM 4656 N LYS B 333 5, .580 -9, .189 28. .589 1. .00 15, .39 N
ATOM 4658 CA LYS B 333 4 .489 -8 .693 27, .748 1, .00 17 .34 C
ATOM 4660 CB LYS B 333 4, .458 -9, .450 26, .441 1. .00 17 .67 C
ATOM 4663 CG LYS B 333 5 .438 -9 .004 25, .401 1, .00 21 .32 C
ATOM 4666 CD LYS B 333 5 .200 -9 .807 24, .128 1. .00 23 .42 C
ATOM 4669 CE LYS B 333 6 .357 -9 .619 23 .113 1. .00 28 .93 C
ATOM 4672 NZ LYS B 333 6 .005 -10 .290 21, .820 1, .00 28 .19 N
ATOM 4676 C LYS B 333 3, .127 -8 .789 28, .452 1, .00 17 .99 C
ATOM 4677 O LYS B 333 2, .338 -7, .868 28, .410 1. .00 15 .95 O
ATOM 4678 N LYS B 334 2, .858 -9, .904 29. .125 1. .00 19, .30 N
ATOM 4680 CA LYS B 334 1 .595 -10 .046 29, .842 1. .00 19 .86 C
ATOM 4682 CB LYS B 334 1 .417 -11. .432 30, .481 1. .00 21 .24 C
ATOM 4685 CG LYS B 334 0, .949 -12, .476 29, .568 1. .00 28, .52 C
ATOM 4688 CD LYS B 334 0, .499 -13, .819 30. .317 1. .00 33, .48 C
ATOM 4691 CE LYS B 334 0 .650 -15 .022 29 .319 1 .00 37 .14 C
ATOM 4694 NZ LYS B 334 0 .870 -14 .604 27. .850 1. .00 36 .97 N
ATOM 4698 C LYS B 334 1 .457 -8 .983 30, .890 1, .00 18 .97 C
ATOM 4699 O LYS B 334 0 .386 -8 .343 31, .021 1, .00 16 .86 O
ATOM 4700 N VAL B 335 2 .522 -8. .754 31, .658 1, ,00 16 .84 N
ATOM 4702 CA VAL B 335 2 .480 -7 .783 32, .726 1, .00 16 .65 C
ATOM 4704 CB VAL B 335 3 .743 -7 .823 33, .591 1, .00 15 .76 C
ATOM 4706 CGI VAL B 335 3, .817 -6. .655 34, ,612 1, .00 19 .24 C
ATOM 4710 CG2 VAL B 335 3, .789 -9, .108 34, ,341 1. ,00 17, .38 C
ATOM 4714 C VAL B 335 2, .288 -6, .382 32. .203 1. .00 15, .76 C
ATOM 4715 O VAL B 335 1 .494 -5 .615 32, .763 1. .00 16 .18 O
ATOM 4716 N ILE B 336 3, .033 -6, .023 31, ,167 1. .00 15 .08 N
ATOM 4718 CA ILE B 336 2 .903 -4 .703 30, .635 1, .00 14 .79 C
ATOM 4720 CB ILE B 336 3 .967 -4 .441 29, .592 1. .00 14 .82 C
ATOM 4722 CGI ILE B 336 5 .290 -4 .318 30 .367 1 .00 16 .31 C
ATOM 4725 CDl ILE B 336 6 .479 -4 .602 29, .579 1, .00 19 .31 C
ATOM 4729 CG2 ILE B 336 3 .543 -3 .266 28, .733 1, .00 16 .34 C
ATOM 4733 C ILE B 336 1 .508 -4 .472 30, .074 1, .00 15 .10 C
ATOM 4734 O ILE B 336 0, .914 -3, .437 30. ,347 1, ,00 16. .57 O
ATOM 4735 N LEU B 337 0 .956 -5 .456 29, ,390 1, .00 15 .14 N
ATOM 4737 CA LEU B 337 0 .343 -5 .235 28, .769 1, .00 15 .94 C
ATOM 4739 CB LEU B 337 0, .645 -6, .290 27. ,727 1, .00 15 .77 C
ATOM 4742 CG LEU B 337 0, .121 -6, .195 26. .404 1. ,00 13, .58 C
ATOM 4744 CDl LEU B 337 0 .021 -7 .498 25. .632 1. .00 15 .44 C
ATOM 4748 CD2 LEU B 337 0 .350 -5 .049 25 .553 1 .00 17 .85 C
ATOM 4752 C LEU B 337 1 .450 -5 .146 29. .810 1, .00 17 .57 C
ATOM 4753 O LEU B 337 2 .511 -4 .531 29, .544 1, .00 17 .36 O
ATOM 4754 N GLN B 338 1 .210 -5 .702 30, .985 1, .00 18 .09 N
ATOM 4756 CA GLN B 338 2 .195 -5 .610 32, .047 1 .00 21 .31 C
ATOM 4758 CB GLN B 338 1 .830 -6 .557 33, .205 1, .00 21 .97 C
ATOM 4761 CG GLN B 338 1 .842 -8 .050 32, .775 1, .00 26 .01 C
ATOM 4764 CD GLN B 338 1 .562 -9 .127 33, .893 1, .00 29 .25 C ATOM 4765 OEl GLN B 338 -0,.720 -8,.942 34..831 1.,00 34..90 o
ATOM 4766 NE2 GLN B 338 -2. .211 -10, .262 33. .738 1. ,00 29, .07 N
ATOM 4769 C GLN B 338 -2. .398 -4, .143 32. .469 1. ,00 22, .67 C
ATOM 4770 O GLN B 338 -3. .530 -3, .694 32. .741 1. ,00 22, .11 O
ATOM 4771 N ASP B 339 -1, .318 -3 .366 32. .465 1. .00 21, .30 N
ATOM 4773 CA ASP B 339 -1. .377 -1, .936 32. .761 1. ,00 21, .98 C
ATOM 4775 CB ASP B 339 -0, ,047 -1, .478 33. .381 1. ,00 22, .85 C
ATOM 4778 CG ASP B 339 0, ,213 -2, .106 34. .710 1. ,00 26, .72 C
ATOM 4779 OD1 ASP B 339 -0, .740 -2 .105 35. .518 1, .00 33 .25 O
ATOM 4780 OD2 ASP B 339 1, .269 -2 .680 35. .026 1, .00 26 .47 O
ATOM 4781 C ASP B 339 -1, .644 -1, .066 31, .555 1. .00 22, .06 C
ATOM 4782 O ASP B 339 -2, .247 0, .004 31. .667 1, .00 23, .60 O
ATOM 4783 N LYS B 340 -1, .218 -1, .515 30. .387 1. .00 19, .19 N
ATOM 4785 CA LYS B 340 -1 .226 -0 .722 29, .218 1. .00 18 .98 C
ATOM 4787 CB LYS B 340 0, .186 -0 .070 29, .118 1. .00 18 .88 C
ATOM 4790 CG LYS B 340 0, .345 0 .901 28, .024 1. .00 18 .59 C
ATOM 4793 CD LYS B 340 1, .805 1 .491 28, .014 1. .00 19, .17 C
ATOM 4796 CE LYS B 340 1, .999 2, .462 26. .848 1. .00 20, .90 C
ATOM 4799 NZ LYS B 340 1 .137 3 .683 27. .041 1. .00 23 .19 N
ATOM 4803 C LYS B 340 -1 .611 -1 .550 28. .035 1. .00 18 .75 C
ATOM 4804 O LYS B 340 -0 .822 -1 .920 27. .224 1. .00 18 .54 O
ATOM 4805 N PRO B 341 -2, .906 -1 .822 27, .892 1. .00 17 .67 N
ATOM 4806 CA PRO B 341 -3 .391 -2 .768 26. .911 1. .00 17 .68 C
ATOM 4808 CB PRO B 341 -4 .926 -2. .753 27, .108 1. ,00 18 .59 C
ATOM 4811 CG PRO B 341 -5 .155 -1 .955 28, .331 1. .00 19 .20 C
ATOM 4814 CD PRO B 341 -3 .919 -1 .347 28, .825 1. .00 20 .06 C
ATOM 4817 C PRO B 341 -3, .101 -2 .444 25, .491 1. .00 15 .31 C
ATOM 4818 O PRO B 341 -3 .022 -3 .289 24 .614 1. ,00 19 .02 O
ATOM 4819 N GLU B 342 -2 .986 -1 .143 25, .254 1. .00 17 .33 N
ATOM 4821 CA GLU B 342 -2 .745 -0 .687 23, .906 1. .00 17 .70 C
ATOM 4823 CB GLU B 342 -3, .615 0 .545 23, .555 1. .00 20 .27 C
ATOM 4826 CG GLU B 342 -5 .028 0 .042 23 .191 1. .00 21 .84 C
ATOM 4829 CD GLU B 342 -6 .029 1 .135 22, .803 1. .00 30 .03 C
ATOM 4830 OEl GLU B 342 -5 .646 2 .108 22, .127 1. .00 34 .62 O
ATOM 4831 OE2 GLU B 342 -7 .217 0 .982 23, .131 1. ,00 29 .42 O
ATOM 4832 C GLU B 342 -1, .232 -0 .491 23, .581 1. .00 17 .99 C
ATOM 4833 O GLU B 342 -0 .912 -0 .071 22 .473 1. .00 17 .45 O
ATOM 4834 N ALA B 343 -0 .348 -0 .902 24. .474 1, ,00 18 .01 N
ATOM 4836 CA ALA B 343 ■ 1 .075 -0 .825 24, .187 1. .00 17 .75 C
ATOM 4838 CB ALA B 343 1 .886 -1 .320 25, .374 1. .00 17 .76 C
ATOM 4842 C ALA B 343 1, .501 -1 .509 22, .934 1. .00 18. .41 C
ATOM 4843 O ALA B 343 1 .018 -2 .612 22 .539 1, ,00 16 .39 O
ATOM 4844 N GLN B 344 2 .398 -0 .808 22 .223 1, .00 16 .93 N
ATOM 4846 CA GLN B 344 3, .007 -1 .326 21, .061 1, ,00 16 .94 C
ATOM 4848 CB GLN B 344 3 .197 -0 .264 19, .951 1. .00 18 .67 C
ATOM 4851 CG GLN B 344 1 .915 0 .367 19 .450 1, .00 22 .45 C
ATOM 4854 CD GLN B 344 1 .041 -0 .668 18 .836 1, .00 23 .74 C
ATOM 4855 OEl GLN B 344 1 .336 -1 .176 17 .737 1, .00 28 .25 O
ATOM 4856 NE2 GLN B 344 0 .022 -1 .076 19, .570 1, .00 24 .17 N
ATOM 4859 C GLN B 344 4 .371 -1 .804 21, .578 1, .00 16 .61 C
ATOM 4860 O GLN B 344 5 .276 -0 .987 21 .833 1, .00 15 .55 O
ATOM 4861 N ILE B 345 4 .503 -3 .109 21 .745 1, .00 14 .81 N
ATOM 4863 CA ILE B 345 5 .747 -3 .697 22. .338 1, .00 15 .57 C
ATOM 4865 CB ILE B 345 5 .426 -4 .864 23, .229 1, .00 14 .52 C
ATOM 4867 CGI ILE B 345 4 .420 -4 .437 24 .307 1, .00 16 .03 C
ATOM 4870 CDl ILE B 345 4 .237 -5 .385 25 .452 1, .00 21 .70 C
ATOM 4874 CG2 ILE B 345 6 .683 -5 .445 23 .832 1, .00 18 .60 C
ATOM 4878 C ILE B 345 6 .713 -4 .108 21, .261 1, .00 15 .60 C
ATOM 4879 O ILE B 345 6 .317 -4 .759 20, .335 1, .00 14 .53 O
ATOM 4880 N ILE B 346 7 .947 -3 .618 21 .338 1, .00 15 .09 N
ATOM 4882 CA ILE B 346 9 .006 -3 .853 20 .373 1, .00 17 .65 C
ATOM 4884 CB ILE B 346 9 .607 -2 .448 19 .887 1, .00 18 .73 C
ATOM 4886 CGI ILE B 346 8 .486 -1 .515 19, .488 1, .00 25 .44 C ATOM 4889 CDl ILE B 346 7,.656 -2,.110 18..400 1..00 27,.09 c
ATOM 4893 CG2 ILE B 346 10. .538 -2. .639 18. .736 1. .00 24, .66 c
ATOM 4897 C ILE B 346 10. .126 -4. .549 21. .136 1. .00 15, .44 c
ATOM 4898 O ILE B 346 10. .515 -4. .048 22. ,174 1. .00 15, .03 o
ATOM 4899 N VAL B 347 10, .621 -5. .685 20. .637 1, .00 15 .30 N
ATOM 4901 CA VAL B 347 11, .701 -6, .425 21. .310 1, .00 14, .26 C
ATOM 4903 CB VAL B 347 11, .363 -7. .916 21. .411 1. .00 13, .95 C
ATOM 4905 CGI VAL B 347 12, ,550 -8. .744 21. .949 1. .00 15, .52 C
ATOM 4909 CG2 VAL B 347 10, .056 -8, .103 22. .171 1, .00 16 .01 C
ATOM 4913 C VAL B 347 12, .980 -6, .253 20. .518 1, .00 13 .34 c
ATOM 4914 O VAL B 347 12, .999 -6, .387 19. .275 1. .00 13. .71 0
ATOM 4915 N LEU B 348 14, ,025 -5, .824 21. .225 1. .00 13, .58 N
ATOM 4917 CA LEU B 348 15, .334 -5. .595 20. ,654 1. .00 14, .50 C
ATOM 4919 CB LEU B 348 15, .629 -4, .101 20. .619 1. .00 14 .73 C
ATOM 4922 CG LEU B 348 14 .624 -3 .255 19, .836 1 .00 18 .66 C
ATOM 4924 CDl LEU B 348 14 .885 -1 .758 20, .172 1. .00 20 .34 C
ATOM 4928 CD2 LEU B 348 14 .781 -3 .515 18, .370 1, .00 21 .48 C
ATOM 4932 C LEU B 348 16. .420 -6, .246 21. .500 1. .00 13 .69 C
ATOM 4933 O LEU B 348 16 .298 -6 .400 22. .715 1 .00 12 .97 o
ATOM 4934 N PRO B 349 17 .533 -6 .626 20. .864 1, .00 13 .80 N
ATOM 4935 CA PRO B 349 18 .630 -7 .173 21, .635 1, .00 12 .71 C
ATOM 4937 CB PRO B 349 19 .686 -7. .528 20, .564 1, .00 14 .22 C
ATOM 4940 CG PRO B 349 18 .940 -7 .614 19 .288 1 .00 17 .12 C
ATOM 4943 CD PRO B 349 17 .802 -6 .586 19, .422 1 .00 13 .86 C
ATOM 4946 C PRO B 349 19 .238 -6 .154 22, .603 1, .00 12 .67 C
ATOM 4947 O PRO B 349 19 .358 -4 .987 22, .270 1, .00 10 .43 0
ATOM 4948 N VAL B 350 19, .679 -6, .629 23, .756 1, .00 10 .88 N
ATOM 4950 CA VAL B 350 20 .463 -5 .844 24, .676 1 .00 11 .84 C
ATOM 4952 CB VAL B 350 20 .967 -6 .740 25, .861 1, .00 12 .85 C
ATOM 4954 CGI VAL B 350 21 .918 -7 .822 25, .385 1, .00 14 .03 C
ATOM 4958 CG2 VAL B 350 21. .614 -5 .822 26, .921 1, .00 13 .28 C
ATOM 4962 C VAL B 350 21 .627 -5 .189 23, .892 1 .00 10 .88 C
ATOM 4963 O VAL B 350 22 .262 -5 .815 23, .000 1 .00 10 .87 O
ATOM 4964 N GLY B 351 21 .864 -3 .926 24, .205 1, .00 11 .86 N
ATOM 4966 CA GLY B 351 22 .882 -3 .099 23, .581 1, .00 11 .51 C
ATOM 4969 C GLY B 351 22 .512 -2 .331 22, .313 1, .00 12 .47 C
ATOM 4970 O GLY B 351 23 .335 -1 .618 21, .744 1. .00 10 .98 O
ATOM 4971 N THR B 352 21 .315 -2 .561 21, .822 1, .00 11 .64 N
ATOM 4973 CA THR B 352 20 .841 -1 .861 20, .642 1, .00 11 .92 C
ATOM 4975 CB THR B 352 19, .508 -2, .419 20, .225 1, .00 13 .71 C
ATOM 4977 OGl THR B 352 19, .641 -3, .812 19. .860 1, ,00 10 .89 O
ATOM 4979 CG2 THR B 352 18 .993 -1 .703 18. .981 1, .00 12 .02 C
ATOM 4983 C THR B 352 20 .720 -0 .383 20, .920 1, .00 11 .24 C
ATOM 4984 O THR B 352 20, .235 0 .015 21. .976 1, .00 12 .17 O
ATOM 4985 N ILE B 353 21, .218 0, .435 20. .012 1, .00 11 .34 N
ATOM 4987 CA ILE B 353 21 .125 1 .893 20, .166 1 .00 11 .58 C
ATOM 4989 CB ILE B 353 22, .322 2 .605 19, .504 1, .00 13 .21 C
ATOM 4991 CGI ILE B 353 23, .642 2, .014 19, .992 1, .00 11 .55 C
ATOM 4994 CDl ILE B 353 23, .795 1 .980 21. .496 1, .00 15 .61 C
ATOM 4998 CG2 ILE B 353 22, .294 4, .098 19. .802 1, .00 13. .36 C
ATOM 5002 C ILE B 353 19 .828 2 .332 19, .509 1, .00 11 .44 C
ATOM 5003 O ILE B 353 19, .485 1 .827 18, .417 1, .00 11 .02 O
ATOM 5004 N VAL B 354 19, .136 3, .268 20, .180 1, .00 10 .73 N
ATOM 5006 CA VAL B 354 17, .785 3, .663 19. .740 1, ,00 11 .42 C
ATOM 5008 CB VAL B 354 16 .693 3 .043 20. .663 1, .00 11 .30 C
ATOM 5010 CGI VAL B 354 16. .741 1 .521 20, .568 1, .00 11 .88 C
ATOM 5014 CG2 VAL B 354 16, .873 3. .542 22, .103 1, .00 12 .49 C
ATOM 5018 C VAL B 354 17, .558 5, .152 19. .702 1, .00 10 .76 C
ATOM 5019 O VAL B 354 18, .289 5, .918 20. .294 1, ,00 11 .15 O
ATOM 5020 N THR B 355 16, .607 5 .599 18, .894 1, .00 10 .94 N
ATOM 5022 CA THR B 355 16 .207 7 .001 18 .884 1. .00 12 .16 C
ATOM 5024 CB THR B 355 15 .004 7 .267 17, .939 1 .00 13 .72 C
ATOM 5026 OGl THR B 355 13 .885 6 .481 18, .413 1 .00 16 .24 O ATOM 5028 CG2 THR B 355 15.313 6.840 16.561 .00 14.33 C
ATOM 5032 C THR B 355 15.684 7.378 20.254 .00 12.37 C
ATOM 5033 O THR B 355 15.263 6.554 21.050 .00 11.43 O
ATOM 5034 N MET B 356 15.787 8.659 20.540 .00 12.52 N
ATOM 5036 CA MET B 356 15.258 9.222 21.775 .00 11.53 C
ATOM 5038 CB MET B 356 16.311 10.075 22.468 .00 11.42 C
ATOM 5041 CG MET B 356 17.427 9.202 23.035 .00 12.95 C
ATOM 5044 SD MET B 356 16.825 8.040 24.219 .00 15.08 S
ATOM 5045 CE MET B 356 17.963 6.799 24.107 .00 20.89 C
ATOM 5049 C MET B 356 13.952 9.961 21.568 .00 13.71 c
ATOM 5050 O MET B 356 13.675 11.024 22.215 .00 12.74 o
ATOM 5051 N GLU B 357 13.148 9.417 20.659 .00 14.50 N
ATOM 5053 CA GLU B 357 11.765 9.869 20.543 .00 14.30 C
ATOM 5055 CB GLU B 357 11.135 9.357 19.210 .00 15.52 C
ATOM 5058 CG GLU B 357 9.674 .735 19.153 .00 15.25 C
ATOM 5061 CD GLU B 357 8.947 .187 17.960 .00 18.46 C
ATOM 5062 OEl GLU B 357 7.768 .559 17.802 .00 19.97 O
ATOM 5063 OE2 GLU B 357 9.543 8.377 17.237 .00 20.59 0
ATOM 5064 C GLU B 357 11.001 9.282 21.703 .00 14.50 c
ATOM 5065 O GLU B 357 11.233 8.123 22.030 .00 14.99 0
ATOM 5066 N TYR B 358 10.129 10.074 22.335 .00 13.93 N
ATOM 5068 CA TYR B 358 9.207 9.612 23.363 .00 14.38 C
ATOM 5070 CB TYR B 358 9.026 10.601 24.499 .00 14.43 C
ATOM 5073 CG TYR B 358 8.057 10.136 25.563 .00 12.84 C
ATOM 5074 CDl TYR B 358 8.436 9.165 26.487 1.00 14.52 C
ATOM 5076 CEl TYR B 358 7.559 8.706 27.463 1.00 19.65 c
ATOM 5078 CZ TYR B 358 .266 9.240 27.504 1.00 19.95 c
ATOM 5079 OH TYR B 358 .321 8.903 28.447 1.00 26.07 o
ATOM 5081 CE2 TYR B 358 ,873 10.184 26.607 1.00 16.96 c
ATOM 5083 CD2 TYR B 358 .767 10.650 25.643 1.00 18.09 c
ATOM 5085 C TYR B 358 7.867 9.326 22.712 1 1. .00 15 .23 c
ATOM 5086 O TYR B 358 7.142 10.261 22.340 1 1 .00 14 .02 o
ATOM 5087 N ARG B 359 .551 8.048 22.606 1 .00 15 .15 N
ATOM 5089 CA ARG B 359 .294 7. 557 22.003 1, .00 16 .88 C
ATOM 5091 CB ARG B 359 .615 6, 465 20.998 1 .00 18 .79 C
ATOM 5094 CG ARG B 359 .824 6.884 19.703 1, .00 22 .92 c
ATOM 5097 CD ARG B 359 .435 5.794 18.692 1 .00 25 .80 c
ATOM 5100 NE ARG B 359 .237 6.083 17.556 1 .00 25 .82 N
ATOM 5102 CZ ARG B 359 .515 5.241 16.593 1, .00 26 .84 C
ATOM 5103 NHl ARG B 359 7.000 3.996 16.593 1, .00 26 .53 N
ATOM 5106 NH2 ARG B 359 8.297 5.668 15.627 1 .00 28 .90 N
ATOM 5109 C ARG B 359 5..449 6.946 23.095 00 17.14 C
ATOM 5110 O ARG B 359 5..767 5.861 23.625 00 15.98 O
ATOM 5111 N ILE B 360 4..401 7.656 23.504 00 16.16 N
ATOM 5113 CA ILE B 360 3, .633 7.262 24.670 00 17.53 C
ATOM 5115 CB ILE B 360 2.575 8.354 24.998 00 17.66 c
ATOM 5117 CGI ILE B 360 895 8.067 26.333 00 24.45 c
ATOM 5120 CDl ILE B 360 711 9.278 27.140 00 28.72 c
ATOM 5124 CG2 ILE B 360 506 8.417 23.930 00 18.76 c
ATOM 5128 C ILE B 360 947 899 24.595 00 16.17 c
ATOM 5129 O ILE B 360 649 307 25.629 00 19.59 0
ATOM 5130 N ASP B 361 692 431 23.402 00 19.19 N
ATOM 5132 CA ASP B 361 049 131 23.290 00 20.04 C
ATOM 5134 CB ASP B 361 070 141 22.156 00 22.33 C
ATOM 5137 CG ASP B 361 0.210 932 22.509 00 27.91 c
ATOM 5138 ODl ASP B 361 0.701 823 23.693 00 33.70 0
ATOM 5139 OD2 ASP B 361 0.734 711 21.687 00 38.62 o
ATOM 5140 C ASP B 361 025 996 23.111 00 19.38 c
ATOM 5141 O ASP B 361 580 872 22.958 00 18.79 0
ATOM 5142 N ARG B 362 342 291 23.099 00 15.34 N
ATOM 5144 CA ARG B 362 342 241 22.870 00 15.87 c
ATOM 5146 CB ARG B 362 6.480 793 21.992 00 13.90 c
ATOM 5149 CG ARG B 362 7.657 839 21.867 00 16.63 c ATOM 5152 CD ARG B 362 8,.570 2.234 20..721 1..00 16.00 c
ATOM 5155 NE ARG B 362 9, .204 3 .494 21. .085 1. .00 15 .40 N
ATOM 5157 CZ ARG B 362 9, .808 4 .315 20. ,234 1. .00 14 .22 C
ATOM 5158 NHl ARG B 362 9, .893 4, .014 18. ,939 1. ,00 16 .86 N
ATOM 5161 NH2 ARG B 362 10. .295 5 .449 20. .686 1, .00 14 .53 N
ATOM 5164 C ARG B 362 5, .970 1 .737 24. .163 1, .00 14 .75 C
ATOM 5165 O ARG B 362 6, .130 2 .504 25. .117 1, .00 13 .39 O
ATOM 5166 N VAL B 363 6, .285 0, .467 24. ,199 1. .00 13 .88 N
ATOM 5168 CA VAL B 363 7, .159 -0 .094 25. .241 1, .00 14 .50 C
ATOM 5170 CB VAL B 363 6, .448 -0 .910 26. .349 1, .00 14 .42 C
ATOM 5172 CGI VAL B 363 7, .477 -1 .351 27. .372 1. .00 17 .68 C
ATOM 5176 CG2 VAL B 363 5, .401 -0, .069 27. .015 1, ,00 16 .62 C
ATOM 5180 C VAL B 363 8, .230 -0, .943 24. ,576 1. ,00 14, .33 C
ATOM 5181 O VAL B 363 7, .928 -1 .989 23. .984 1, .00 14 .74 O
ATOM 5182 N ARG B 364 9 .483 -0 .510 24. .651 1 .00 13 .57 N
ATOM 5184 CA ARG B 364 10 .570 -1 .349 24. .131 1 .00 13 .59 C
ATOM 5186 CB ARG B 364 11 .753 -0 .458 23. .713 1 .00 12 .88 C
ATOM 5189 CG ARG B 364 11 .565 0 .380 22. .482 1, .00 14 .21 C
ATOM 5192 CD ARG B 364 12 .671 1 .411 22, .216 1 .00 18 .75 C
ATOM 5195 NE ARG B 364 12 .606 2 .066 20, .950 1 .00 16 .44 N
ATOM 5197 CZ ARG B 364 13 .123 3 .278 20, .638 1 .00 18 .34 C
ATOM 5198 NHl ARG B 364 13 .591 4 .073 21. .531 1, .00 21 .95 N
ATOM 5201 NH2 ARG B 364 13 .073 3 .670 19, .412 1 .00 23 .97 N
ATOM 5204 C ARG B 364 11 .046 -2 .339 25, .177 1 .00 14 .09 C
ATOM 5205 O ARG B 364 11 .154 -1 .994 26, .361 1 .00 16 .64 O
ATOM 5206 N LEU B 365 11 .340 -3 .548 24. .735 1, .00 13 .84 N
ATOM 5208 CA LEU B 365 11 .904 -4 .519 25. .617 1, .00 12 .64 C
ATOM 5210 CB LEU B 365 11 .017 -5 .737 25. .667 1 .00 14 .17 C
ATOM 5213 CG LEU B 365 9 .653 -5 .571 26, .305 1 .00 14 .30 C
ATOM 5215 CDl LEU B 365 8 .907 -6 .860 26, .267 1, .00 17 .46 C
ATOM 5219 CD2 LEU B 365 9 .813 -5 .054 27. .714 1, .00 18 .97 C
ATOM 5223 C LEU B 365 13 .235 -4 .929 25, .065 1 .00 13 .48 C
ATOM 5224 O LEU B 365 13 .307 -5 .386 23, .919 1 .00 13 .99 O
ATOM 5225 N PHE B 366 14 .271 -4 .775 25, .887 1, .00 12 .70 N
ATOM 5227 CA PHE B 366 15, .619 -5 .176 25. .533 1, .00 1 .04 C
ATOM 5229 CB PHE B 366 16, .651 -4 .148 25. .971 1, .00 12 .03 C
ATOM 5232 CG PHE B 366 16 .476 -2 .809 25. .291 1 .00 13 .71 C
ATOM 5233 CDl PHE B 366 15 .591 -1 .889 25. .820 1, .00 13 .42 c
ATOM 5235 CEl PHE B 366 15, .373 -0 .702 25. .229 1, .00 16 .78 c
ATOM 5237 CZ PHE B 366 16, .025 -0 .376 24. .078 1, .00 16 .52 c
ATOM 5239 CE2 PHE B 366 16, .899 -1, .322 23. .444 1, .00 15 .87 c
ATOM 5241 CD2 PHE B 366 17 .121 -2 .554 24, .083 1, .00 15 .60 c
ATOM 5243 C PHE B 366 15 .875 -6 .502 26, .202 1, .00 13 .03 c
ATOM 5244 O PHE B 366 15 .700 -6 .682 27, .407 1, .00 13 .72 0
ATOM 5245 N VAL B 367 16, .319 -7 .448 25. .407 1, .00 13 .72 N
ATOM 5247 CA VAL B 367 16 .457 -8 .824 25, .906 1 .00 14 .35 C
ATOM 5249 CB VAL B 367 15 .408 -9 .719 25, .263 1, .00 15 .42 C
ATOM 5251 CGI VAL B 367 13. .988 -9 .325 25. .626 1, .00 17 .97 C
ATOM 5255 CG2 VAL B 367 15, .608 -9 .853 23. .736 1, .00 13 .87 c
ATOM 5259 C VAL B 367 17, .829 -9, .418 25. .654 1. .00 14 .47 c
ATOM 5260 O VAL B 367 18 .508 -9 .099 24, .686 1, .00 13 .82 0
ATOM 5261 N ASP B 368 18, .186 -10 .385 26. .500 1, .00 15 .21 N
ATOM 5263 CA ASP B 368 19, ,421 -11 .145 26. .337 1. .00 14 .26 C
ATOM 5265 CB ASP B 368 20, .020 -11 .526 27. .668 1. .00 12 .83 C
ATOM 5268 CG ASP B 368 19 .168 -12 .492 28, .461 1. .00 13 .78 C
ATOM 5269 ODl ASP B 368 18 .383 -13 .277 27. .864 1, .00 15 .03 O
ATOM 5270 OD2 ASP B 368 19, .284 -12 .487 29. .687 1. .00 14 .11 0
ATOM 5271 C ASP B 368 19, .197 -12 .347 25. .438 1, .00 15 .37 c
ATOM 5272 O ASP B 368 18, .090 -12, .572 24. .940 1. .00 13 ,77 o
ATOM 5273 N LYS B 369 20 .201 -13 .219 25, .295 1, .00 15 .53 N
ATOM 5275 CA LYS B 369 20 .096 -14 .274 24, .320 1 .00 16 .17 C
ATOM 5277 CB LYS B 369 21 .492 -14 .913 24, .031 1 .00 18 .57 C
ATOM 5280 CG LYS B 369 22 .335 -14 .040 23, .132 1. .00 23 .43 C ATOM 5283 CD LYS B 369 21.692 -13.930 21.723 1.00 33.33 c
ATOM 5286 CE LYS B 369 21 .825 -15 .245 20 .863 1. .00 37 .24 c
ATOM 5289 NZ LYS B 369 20 .834 -15 .304 19 .703 1. .00 39 .15 N
ATOM 5293 C LYS B 369 19 .120 -15 .342 24 .746 1. .00 17 .09 C
ATOM 5294 O LYS B 369 18 .736 -16 .166 23 .914 1 .00 16 .83 O
ATOM 5295 N LEU B 370 18 .744 -15 .352 26 .029 1, .00 15 .51 N
ATOM 5297 CA LEU B 370 17 .739 -16 .298 26 .509 1, .00 15 .40 C
ATOM 5299 CB LEU B 370 18 .053 -16 .814 27, .899 1, .00 15 .53 C
ATOM 5302 CG LEU B 370 19 .358 -17 .657 28 .054 1, .00 17 .89 C
ATOM 5304 CDl LEU B 370 19 .624 -17 .980 29 .490 1, .00 16 .28 C
ATOM 5308 CD2 LEU B 370 19 .237 -18 .944 27 .314 1, .00 17 .77 C
ATOM 5312 C LEU B 370 16, .346 -15 .711 26, .481 1, .00 17, .02 C
ATOM 5313 O LEU B 370 15, .426 -16 .340 27, .005 1. .00 16, .78 O
ATOM 5314 N ASP B 371 16 .191 -14 .538 25 .884 1, .00 15 .62 N
ATOM 5316 CA ASP B 371 14 .909 -13 .828 25 .822 1 .00 17 .82 C
ATOM 5318 CB ASP B 371 13 .844 -14 .610 25 .070 1, .00 16 .70 C
ATOM 5321 CG ASP B 371 13 .943 -14 .455 23 .561 1, .00 26 .05 C
ATOM 5322 ODl ASP B 371 14 .579 -13 .492 23, .026 1. .00 26 .78 O
ATOM 5323 OD2 ASP B 371 13 .333 -15 .252 22 .835 1, .00 35 .44 O
ATOM 5324 C ASP B 371 14 .392 -13 .390 27. .197 1, .00 16 .31 C
ATOM 5325 O ASP B 371 13, .179 -13 .251 27, .432 1. .00 15 .98 O
ATOM 5326 N ASN B 372 15, .334 -13 .152 28, .115 1. .00 14, .12 N
ATOM 5328 CA ASN B 372 15 .057 -12 .509 29 .380 1. .00 13 .45 C
ATOM 5330 CB ASN B 372 15 .846 -13 .137 30, .518 1, .00 14 .25 C
ATOM 5333 CG ASN B 372 15, .359 -14 .530 30, .868 1. .00 17 .78 c
ATOM 5334 ODl ASN B 372 14, .170 -14 .830 30, .726 1. .00 15, .50 O
ATOM 5335 ND2 ASN B 372 16, .280 -15 .394 31, .268 1. .00 15, .27 N
ATOM 5338 C ASN B 372 15 .328 -11 .025 29, .328 1. .00 13 .56 C
ATOM 5339 O ASN B 372 16, .185 -10 .535 28, .526 1. .00 12, .51 O
ATOM 5340 N ILE B 373 14 .521 -10 .271 30, .078 1. .00 12 .00 N
ATOM 5342 CA ILE B 373 14, .595 -8 .793 30, .098 1. .00 12 .80 C
ATOM 5344 CB ILE B 373 13 .409 -8 .177 30 .868 1, .00 12 .48 C
ATOM 5346 CGI ILE B 373 12 .115 -8 .624 30, .263 1. .00 14 .38 C
ATOM 5349 CDl ILE B 373 12, .021 -8 .386 28, .913 1. .00 13 .59 C
ATOM 5353 CG2 ILE B 373 13, .467 -6 .685 30, .862 1. .00 13, .18 C
ATOM 5357 C ILE B 373 15, .888 -8. .319 30. .716 1. ,00 12, .70 C
ATOM 5358 O ILE B 373 16, .217 -8 .667 31. .872 1. .00 13 .25 O
ATOM 5359 N ALA B 374 16, .619 -7 .490 29, .940 1. .00 12, .49 N
ATOM 5361 CA ALA B 374 17, .962 -7, .065 30, .334 1. .00 11, .98 C
ATOM 5363 CB ALA B 374 18, ,894 -6, .974 29. .099 1. ,00 11, .19 C
ATOM 5367 C ALA B 374 18. .005 -5, .723 31. .031 1. ,00 12 , .88 C
ATOM 5368 O ALA B 374 19, .017 -5 .394 31. .640 1. .00 13, .61 O
ATOM 5369 N GLU B 375 16, .957 -4. .922 30. .954 1. .00 12, .63 N
ATOM 5371 CA GLU B 375 16, ,927 -3, .628 31. .592 1. ,00 11, .71 C
ATOM 5373 CB GLU B 375 17, .669 -2, .571 30. .740 1. .00 13 , .04 C
ATOM 5376 CG GLU B 375 17 .020 -2 .247 29 .443 1, .00 13 .36 C
ATOM 5379 CD GLU B 375 17 .854 -1 .238 28, .611 1. .00 16 .47 C
ATOM 5380 OEl GLU B 375 18, .961 -1 .591 28, .279 1. .00 19, .52 O
ATOM 5381 OE2 GLU B 375 17, .355 -0 .152 28, .242 1. .00 18, .89 O
ATOM 5382 C GLU B 375 15, .464 -3, .195 31. .858 1. .00 12, .84 c
ATOM 5383 O GLU B 375 14 .563 -3 .750 31, .276 1, .00 12 .27 O
ATOM 5384 N VAL B 376 15, .277 -2 .235 32, .747 1. .00 12. .90 N
ATOM 5386 CA VAL B 376 13, .918 -1 .872 33, .175 1. .00 13, .25 C
ATOM 5388 CB VAL B 376 13, .941 -0, .719 34. .173 1. .00 13, .63 C
ATOM 5390 CGI VAL B 376 12, .515 -0. .240 34. .514 1. .00 15 .72 C
ATOM 5394 CG2 VAL B 376 14, .681 -1 .129 35, .424 1. .00 19, .49 C
ATOM 5398 C VAL B 376 13, .110 -1, .441 32, .018 1. .00 12, .36 C
ATOM 5399 O VAL B 376 13, .458 -0, .494 31. .358 1. ,00 12, .35 o
ATOM 5400 N PRO B 377 12, .016 -2, .117 31. .700 1. ,00 13 , .27 N
ATOM 5401 CA PRO B 377 11, .169 -1 .588 30. .649 1. .00 14, .34 c
ATOM 5403 CB PRO B 377 10 .141 -2 .697 30, .437 1. .00 15 .95 c
ATOM 5406 CG PRO B 377 10 .777 -3 .950 31, .039 1. .00 13 .76 c
ATOM 5409 CD PRO B 377 11, .585 -3 .453 32, .170 1. .00 13 .52 c ATOM 5412 C PRO B 377 10.,472 -0,.302 31.,046 1..00 14..46 c
ATOM 5413 O PRO B 377 10. ,078 -0. .158 32. ,190 1. ,00 14. .50 o
ATOM 5414 N ARG B 378 10. ,291 0 .603 30. .087 1. .00 14. .99 N
ATOM 5416 CA ARG B 378 9. .687 1. .912 30. .298 1. .00 14. .79 C
ATOM 5418 CB ARG B 378 10. .756 3 .004 30. .386 1. .00 14 .41 C
ATOM 5421 CG ARG B 378 11, .717 2 .809 31. .542 1, .00 16, .49 C
ATOM 5424 CD ARG B 378 12. .848 3 .892 31. .600 1, .00 17, .97 C
ATOM 5427 NE ARG B 378 13. .805 3 .569 32. .642 1. .00 15, .14 N
ATOM 5429 CZ ARG B 378 13, .602 3 .673 33, .949 1, .00 19 .35 C
ATOM 5430 NHl ARG B 378 12, .452 4 .173 34. .445 1. .00 21. .30 N
ATOM 5433 NH2 ARG B 378 14, ,548 3. .250 34. .752 1. .00 20, .05 N
ATOM 5436 C ARG B 378 8, .797 2 .263 29. .129 1. .00 14, .34 C
ATOM 5437 O ARG B 378 8, .974 1 .810 28. .013 1. ,00 14, .31 O
ATOM 5438 N VAL B 379 7, .801 3 .052 29. .411 1, .00 13 .60 N
ATOM 5440 CA VAL B 379 7, .028 3 .650 28. .358 1. .00 14, .99 C
ATOM 5442 CB VAL B 379 5, ,847 4 .399 28. .967 1. .00 16, .05 C
ATOM 5444 CGI VAL B 379 5, .148 5 .163 27. .928 1. .00 17, .97 C
ATOM 5448 CG2 VAL B 379 4, .863 3 .410 29. .606 1. ,00 19, .07 C
ATOM 5452 C VAL B 379 7, .899 4 .629 27. .551 1. .00 13, .40 C
ATOM 5453 O VAL B 379 8, .683 5 .397 28. .127 1. .00 13, .64 O
ATOM 5454 N GLY B 380 7, ,742 4 .666 26. .241 1. ,00 13, .02 N
ATOM 5456 CA GLY B 380 8, ,480 5 .637 25. .459 1. ,00 14, .94 C
ATOM 5459 C GLY B 380 8, .968 5 .181 24. .107 1. .00 13 .89 C
ATOM 5460 O GLY B 380 8, .939 3 .974 23. .839 1. .00 13, .82 O
ATOM 5461 OXT GLY B 380 9, .391 6 .068 23. .366 1. ,00 15, .44 O
ATOM 5462 O HOH W 1 26, .337 16 .956 29. .710 1. .00 8 .65 O
ATOM 5465 O HOH W 2 9, .939 2 .105 25. .538 1. .00 12, .99 O
ATOM 5468 O HOH W 3 22 .328 10 .101 24, .400 1. .00 12 .55 O
ATOM 5471 O HOH W 4 30, .572 18 .292 23, .118 1, .00 9 .33 O
ATOM 5474 O HOH W 5 8, .147 23 .150 18. .782 1. .00 14, .00 O
ATOM 5477 O HOH W 6 11, ,956 29 .794 31. .575 1. .00 15, .66 O
ATOM 5480 O HOH W 7 36 .742 23 .674 17, .265 1. .00 14 .06 O
ATOM 5483 O HOH W 8 26. .462 19 .745 37, .226 1. .00 12 .03 O
ATOM 5486 O HOH W 9 23, .101 0 .721 12. .656 1. .00 12, .19 O
ATOM 5489 O HOH W 10 20, .065 -2 .650 26. .156 1. .00 20, .32 O
ATOM 5492 O HOH W 11 18, .435 36 .223 15. .049 1. .00 20, .55 0
ATOM 5495 O HOH W 12 18, .961 -14 .287 31. .415 1, .00 12 .28 0
ATOM 5498 0 HOH w 13 13, .655 -3 .630 28, .503 1. .00 11. .25 o
ATOM 5501 0 HOH w 14 6, .772 28 .494 24. .564 1. .00 16, .02 o
ATOM 5504 0 HOH w 15 25, ,827 -0 .949 23. .096 1. .00 13, .13 o
ATOM 5507 0 HOH w 16 10, ,548 23 .630 17. .206 1. .00 15, .05 o
ATOM 5510 0 HOH w 17 21, .366 -0 .008 27. .896 1, .00 14 .00 o
ATOM 5513 0 HOH w 18 6, .571 29 .390 22, .100 1. .00 18, .38 0
ATOM 5516 0 HOH w 19 25, .418 4 .779 24. .010 1. .00 11, .22 o
ATOM 5519 0 HOH w 20 15, ,446 15 .813 32. .417 1. .00 13, .39 o
ATOM 5522 0 HOH w 21 5 .625 22 .360 17, .718 1, .00 14 .91 o
ATOM 5525 0 HOH w 22 27, .953 5 .617 25, .060 1. .00 12 . 69 o
ATOM 5528 0 HOH w 23 13, .200 17 .441 16. .128 1. .00 12, .98 0
ATOM 5531 0 HOH w 24 42, ,359 19 .143 18. .719 1. .00 14, .71 o
ATOM 5534 0 HOH w 25 24 .537 -2 .025 19, .216 1, .00 12 .97 o
ATOM 5537 0 HOH w 26 27 .926 25 .732 6 .249 1. .00 17 .25 o
ATOM 5540 0 HOH w 27 39 .025 23 .474 22, .653 1, .00 12 .82 0
ATOM 5543 0 HOH w 28 23 .815 15 .465 37, .744 1, .00 13 .10 o
ATOM 5546 0 HOH w 29 18 .367 -9 .817 33, .092 1, ,00 17 .61 o
ATOM 5549 0 HOH w 30 20 .380 12 .554 15, .652 1, .00 10 .66 o
ATOM 5552 0 HOH w 31 18 .651 1 .596 26, .271 1, .00 14 .85 o
ATOM 5555 0 HOH w 32 35 .209 6 .007 10, .838 1, .00 15 .93 o
ATOM 5558 0 HOH w 33 18 .465 24 .874 35, .632 1, ,00 11 .70 o
ATOM 5561 0 HOH w 34 20 .815 27 .470 36, .539 1, .00 14 .90 o
ATOM 5564 0 HOH 35 20 .733 10 .911 9, .565 1, ,00 14 .64 0
ATOM 5567 0 HOH w 36 4 .788 27 .744 28, .587 1, .00 16 .40 o
ATOM 5570 0 HOH w 37 8 .972 17 .007 35, .207 1, .00 15 .88 o
ATOM 5573 0 HOH w 38 33 .433 11 .882 27, .279 1, ,00 15 .71 o ATOM 5576 O HOH W 39 11..974 0..247 27..662 1..00 14,.69 o
ATOM 5579 O HOH W 40 11. .026 22 .081 14, .883 1. .00 14, .23 o
ATOM 5582 O HOH W 41 26, .884 -0, .716 26, .776 1. .00 14, .35 0
ATOM 5585 O HOH W 42 41. ,266 13, .179 18. .993 1. .00 17. .71 o
ATOM 5588 O HOH W 43 27 .981 -2 .861 17 .044 1. .00 20 .05 0
ATOM 5591 O HOH W 44 2, .212 -4 .670 21, .196 1. .00 17, .41 o
ATOM 5594 O HOH W 45 5, ,416 16, .195 31, .844 1. .00 15, .02 0
ATOM 5597 O HOH W 46 20, .229 32 .354 26, .988 1. .00 15, .42 o
ATOM 5600 O HOH W 47 27, .214 14 .437 31, .277 1, .00 10 .24 0
ATOM 5603 O HOH W 48 24, .332 32, .917 12, .832 1. .00 16, .80 o
ATOM 5606 O HOH W 49 9, .986 5 .426 33. .075 1. .00 16, .06 0
ATOM 5609 O HOH W 50 21, .134 30 .372 36, .728 1. .00 15, .50 0
ATOM 5612 O HOH W 51 4. .815 20, .991 24. .941 1. .00 17. .09 0
ATOM 5615 O HOH 52 39, .195 8 .284 21, .866 1. .00 17. .72 o
ATOM 5618 O HOH W 53 24, .661 0 .260 25, .378 1. .00 13 .02 o
ATOM 5621 O HOH W 54 6, .599 11 .219 19, .732 1. .00 15, .01 o
ATOM 5624 O HOH W 55 -1, .402 -3, .742 22. .540 1. .00 17, ,94 0
ATOM 5627 O HOH W 56 23, .967 19 .287 36. .011 1. .00 12 .16 0
ATOM 5630 0 HOH W 57 32, .403 10 .495 30. .395 1. .00 18, ,72 0
ATOM 5633 0 HOH W 58 30, .411 20 .433 3, .925 1. .00 19, .29 0
ATOM 5636 0 HOH W 59 14, .110 5 .986 23. .754 1. .00 21, ,44 0
ATOM 5639 0 HOH W 60 9, .502 31 .255 31, .238 1. .00 19, .33 0
ATOM 5642 0 HOH W 61 17, .881 7 .531 29, .614 1. .00 18, .16 0
ATOM 5645 0 HOH W 62 35 .920 21 .100 27 .416 1, .00 18 .14 0
ATOM 5648 0 HOH w 63 21, .184 17 .585 43, .689 1, .00 19, .63 0
ATOM 5651 0 HOH w 64 11, ,422 6, .357 17. .386 1. .00 17, .47 0
ATOM 5654 0 HOH w 65 7, .568 24 .486 1, .717 1, .00 26, .99 0
ATOM 5657 0 HOH w 66 11 .835 23 .396 12 .551 1, .00 17. .33 0
ATOM 5660 0 HOH w 67 15, .674 -0 .267 13, .102 1. .00 28, .14 0
ATOM 5663 0 HOH w 68 35 .246 4 .205 18, .695 1, .00 18 .92 0
ATOM 5666 0 HOH w 69 12, .071 7 .219 35, .891 1. .00 17, .95 0
ATOM 5669 0 HOH w 70 33 .151 1 .122 14, .747 1. .00 19, .80 0
ATOM 5672 0 HOH w 71 22 .406 17 .015 36 .075 1. .00 12 .42 0
ATOM 5675 0 HOH w 72 20, .744 5 .444 32, .336 1, .00 24, .35 0
ATOM 5678 0 HOH w 73 20, .988 -0, .601 11, .337 1. .00 17, .02 o
ATOM 5681 0 HOH w 74 32, .168 21 .857 32, .986 1. .00 16, .30 o
ATOM 5684 0 HOH w 75 28 .340 34 .388 18. .531 1, .00 14 .67 0
ATOM 5687 0 HOH w 76 27, .395 34 .930 22, .269 1. .00 15, .72 o
ATOM 5690 0 HOH w 77 -2 .881 1 .257 27, .203 1, .00 24 .36 0
ATOM 5693 0 HOH w 78 1, .320 22 .974 26, .514 1, .00 16, .10 o
ATOM 5696 0 HOH w 79 20, .014 2, .049 4. .590 1. .00 18 .49 0
ATOM 5699 0 HOH w 80 20. .131 -10 .521 22, .803 1. .00 16, .67 o
ATOM 5702 0 HOH w 81 2 .801 -12 .938 26, .738 1, .00 21 .33 0
ATOM 5705 0 HOH w 82 13. .524 14 .422 42, .122 1, .00 22, .70 0
ATOM 5708 0 HOH w 83 41. .288 13. .787 8. .384 1. .00 21, ,34 0
ATOM 5711 0 HOH w 84 33 .383 31 .044 25 .382 1 .00 20 .83 0
ATOM 5714 0 HOH w 85 39 .402 29 .928 19, .487 1, .00 19 .25 0
ATOM 5717 0 HOH w 86 14, .181 -0 .702 28, .733 1. .00 20, .17 o
ATOM 5720 0 HOH w 87 11 .217 5 .903 27, .361 1. .00 18 .74 0
ATOM 5723 0 HOH w 88 28, .627 31 .304 33, .345 1. .00 18, .56 0
ATOM 5726 0 HOH w 89 25 .546 35 .322 19, .001 1, .00 21 .03 0
ATOM 5729 0 HOH w 90 17 .693 -0 .865 33. .818 1, .00 23 .45 o
ATOM 5732 0 HOH w 91 16 .853 2 .781 26, .050 1, .00 20 .34 0
ATOM 5735 0 HOH w 92 34, .612 21 .168 14, .066 1, .00 15, .33 o
ATOM 5738 0 HOH w 93 19 .619 19 .119 0 .936 1 .00 29 .35 o
ATOM 5741 0 HOH w 94 20 .721 21 .727 42, .621 1, .00 20 .03 o
ATOM 5744 0 HOH w 95 17 .040 19 .477 42, .629 1, .00 20, .78 o
ATOM 5747 0 HOH w 96 20 .111 -0 .122 24, .735 1, .00 19 .14 o
ATOM 5750 0 HOH w 97 3 .609 25 .498 17, ,596 1, .00 22, .36 o
ATOM 5753 0 HOH w 98 22 .201 34 .610 13, ,464 1. .00 19 .68 o
ATOM 5756 0 HOH w 99 1 .306 27 .285 34 .611 1 .00 28 .89 0
ATOM 5759 0 HOH w 100 1 .618 26 .901 31, .789 1 .00 16 .56 o
ATOM 5762 0 HOH w 101 34 .765 7 .143 28, .884 1, .00 25 .03 0 ATOM 5765 O HOH W 102 39..459 5.374 23,.644 1.00 21.05 o
ATOM 5768 O HOH W 103 7, .666 6 .005 10, .390 1 .00 23. .68 o
ATOM 5771 O HOH W 104 25. .629 6 .850 39, ,327 1, .00 25. .93 0
ATOM 5774 O HOH W 105 4. ,516 10, .592 22. ,714 1, .00 16, .32 o
ATOM 5777 O HOH W 106 23, .065 -8 .147 22, .078 1 .00 22 .91 o
ATOM 5780 O HOH W 107 15, .215 5 .229 8, .027 1. .00 18 .64 0
ATOM 5783 O HOH W 108 9, ,120 -7 .084 18, ,432 1, .00 24, .86 o
ATOM 5786 O HOH W 109 9, ,059 32, .376 26. .670 1, .00 22, .20 0
ATOM 5789 O HOH W 110 28 .414 12 .755 0, .995 1 .00 20 .29 o
ATOM 5792 O HOH W 111 14 .310 19 .153 -3, .544 1 .00 25 .42 0
ATOM 5795 O HOH W 112 18 .007 -11 .145 20, .744 1 .00 25 .31 0
ATOM 5798 O HOH W 113 38, .102 21 .625 11, .695 1 .00 21 .89 0
ATOM 5801 O HOH W 114 6, .120 13 .736 30, .142 1, .00 24. .46 o
ATOM 5804 O HOH W 115 15 .906 -18 .386 31, .627 1 .00 22 .15 o
ATOM 5807 O HOH W 116 22, .389 33 .907 19, .142 1 .00 22 .92 o
ATOM 5810 O HOH W 117 32, .200 35 .724 21, .334 1 .00 26 .32 o
ATOM 5813 O HOH W 118 2, .190 17, .510 6. .678 1, .00 25, .22 0
ATOM 5816 O HOH W 119 1, ,118 4, .704 29. .359 1, .00 25, .72 o
ATOM 5819 O HOH W 120 9, .611 1 .556 17. .312 1 .00 23. .94 o
ATOM 5822 O HOH W 121 3, .629 23, .370 16. .136 1, .00 22, .93 0
ATOM 5825 O HOH W 122 32 .907 25 .232 8, .564 1 .00 22 .34 o
ATOM 5828 O HOH W 123 -1, .806 -9 .230 29. .885 1, .00 26 .14 o
ATOM 5831 O HOH W 124 32 .230 21 .567 30, .291 1 .00 23 .29 o
ATOM 5834 O HOH W 125 37 .450 14 .678 29, .329 1 .00 25 .24 o
ATOM 5837 O HOH w 126 15, .339 36 .760 18, .883 1, .00 20 .61 0
ATOM 5840 0 HOH w 127 10, .235 7 .567 31. .063 1, .00 21, .51 0
ATOM 5843 0 HOH w 128 24, ,897 37, .442 19. .549 1, .00 24, .86 o
ATOM 5846 0 HOH w 129 17 ,030 -13, .244 22, .390 1. .00 21 .48 o
ATOM 5849 0 HOH w 130 9, .040 8 .412 14. ,673 1, .00 27, .72 0
ATOM 5852 0 HOH w 131 14, ,720 31, .661 34. .320 1, .00 22, .71 o
ATOM 5855 0 HOH w 132 19. .535 8, .925 0. .482 1. .00 21, .18 o
ATOM 5858 0 HOH w 133 12 .077 0 .962 18, .542 1 .00 22 .23 o
ATOM 5861 0 HOH w 134 3, .441 12 .279 24, .403 1 .00 21 .97 o
ATOM 5864 0 HOH w 135 13. ,235 21 .344 -4, .722 1, .00 25 .46 o
ATOM 5867 0 HOH w 136 4, .989 14 .655 27, ,582 1, .00 28, .75 0
ATOM 5870 0 HOH w 137 -0, ,388 24, .768 31. .623 1, .00 23, .62 o
ATOM 5873 0 HOH w 138 9 .733 10 .653 39. .989 1, .00 28, .29 o
ATOM 5876 0 HOH w 139 5, .022 2 .800 18, .295 1, .00 34, .14 o
ATOM 5879 0 HOH w 140 26, .430 -3, .864 19. .091 1, .00 26, .57 o
ATOM 5882 0 HOH w 141 33, ,127 12, .459 35. .197 1, .00 24, .55 o
ATOM 5885 0 HOH w 142 4. ,529 14, .653 24. .602 1. .00 27, .79 0
ATOM 5888 0 HOH w 143 34. .889 3, .006 29. .679 1, .00 26, .45 o
ATOM 5891 0 HOH w 144 26, ,472 27, .781 5. .265 1, .00 24, .30 0
ATOM 5894 0 HOH w 145 9 .844 1 .973 10. .352 1, .00 30 .48 o
ATOM 5897 0 HOH w 146 23, .113 35 .988 15, .667 1, .00 24, .26 o
ATOM 5900 0 HOH w 147 3 .506 18 .934 26, .329 1 .00 23 .36 o
ATOM 5903 0 HOH w 148 41, .932 12 .669 10, .942 1, .00 26, .60 o
ATOM 5906 0 HOH w 149 40, .619 12 .671 22, ,354 1, .00 26, .08 0
ATOM 5909 0 HOH w 150 33, ,062 38, .217 15, .479 1, .00 37, .76 o
ATOM 5912 0 HOH w 151 3, ,554 31, .071 10, .635 1. .00 26, .96 o
ATOM 5915 0 HOH w 152 14, .084 8 .928 41, .767 1. .00 28, .97 0
ATOM 5918 0 HOH w 153 29, .827 1, .222 9. .591 1, .00 30, .85 o
ATOM 5921 0 HOH w 154 23, ,088 -1, .722 26. .918 1. .00 27, .80 o
ATOM 5924 0 HOH w 155 8. ,435 8, .580 36. .298 1. .00 27. .72 o
ATOM 5927 0 HOH w 156 42. .926 15, .621 8. .285 1, .00 29. .85 o
ATOM 5930 0 HOH w 157 6 .654 11 .279 36, .797 1 .00 28 .44 o
ATOM 5933 0 HOH w 158 15, .300 6 .642 27, .237 1, .00 23, .08 o
ATOM 5936 0 HOH w 159 14, .085 -11. .817 20, ,799 1. .00 27, .86 o
ATOM 5939 0 HOH w 160 -1, .521 -6, .774 36. .408 1. .00 31, .93 o
ATOM 5942 0 HOH w 161 15 .519 1 .708 31, ,201 1, .00 27, .02 o
ATOM 5945 0 HOH w 162 0, .621 -5 .366 35, .349 1, .00 28, .46 o
ATOM 5948 0 HOH w 163 18, .036 -12 , .751 33. .648 1, .00 22, .09 o
ATOM 5951 0 HOH w 164 32, ,843 18, .669 30. .344 1, .00 28, .48 o ATOM 5954 O HOH W 165 27,,765 11..874 38..295 1..00 18,.10 0
ATOM 5957 O HOH W 166 1, ,781 17. ,606 13. .084 1. .00 28, .85 o
ATOM 5960 O HOH W 167 20, .211 0. .158 6. .249 1. .00 29, .98 o
ATOM 5963 O HOH W 168 2. .759 19. .112 32, ,488 1. .00 24. .07 o
ATOM 5966 O HOH W 169 33. .968 18, .793 32. .524 1. .00 23, .54 o
ATOM 5969 O HOH W 170 -1, ,571 -13, .592 26. .165 1. .00 34, .45 0
ATOM 5972 O HOH W 171 39. ,370 28, .627 17. ,071 1. .00 26, .29 o
ATOM 5975 O HOH W 172 17. .376 32, ,794 35. ,436 1. .00 23, .98 0
ATOM 5978 O HOH W 173 9, .391 6, .761 35. .029 1, ,00 21 .74 0
ATOM 5981 O HOH W 174 16, .352 11, .687 43. .877 1. .00 31, .51 o
ATOM 5984 O HOH W 175 36, .018 4, .292 25. .853 1. .00 21, .19 o
ATOM 5987 O HOH W 176 24, .899 -2, .518 10. .289 1. .00 21, .66 0
ATOM 5990 O HOH W 177 -1, .286 2, .934 25. ,647 1. .00 31, .88 0
ATOM 5993 O HOH W 178 13 .449 -16 .540 28. .665 1, ,00 26 .26 o
ATOM 5996 O HOH W 179 13, .301 -0 .613 16. .357 1, .00 27 .05 o
ATOM 5999 O HOH W 180 24, .842 17, .585 44. .930 1. .00 26, .85 o
ATOM 6002 O HOH W 181 5, .856 18, .874 39. .008 1. .00 29, .19 o
ATOM 6005 O HOH W 182 -1, .630 2, .543 31. .697 1. .00 33, .70 0
ATOM 6008 O HOH W 183 38 .130 17 .164 1. .491 1, .00 33 .90 o
ATOM 6011 O HOH W 184 38, .533 33 .710 21. .252 1, .00 23 .89 0
ATOM 6014 O HOH W 185 8, .687 18, .331 1. .042 1. .00 28, .46 o
ATOM 6017 O HOH W 186 13, .162 5, .211 37. .558 1. .00 29, .88 o
ATOM 6020 O HOH W 187 13 .148 -16 .170 33, .001 1. .00 24 .01 o
ATOM 6023 0 HOH W 188 17 .877 11 .059 1. .344 1. .00 30 .31 0
ATOM 6026 0 HOH w 189 1, .036 -11, .099 25. .828 1, .00 27 .54 o
ATOM 6029 0 HOH w 190 19, .608 24, .676 5. .693 1, .00 30 .78 o
ATOM 6032 0 HOH w 191 19, .946 19, .409 42. .111 1, .00 26, .14 o
ATOM 6035 0 HOH w 192 3 .476 10 .629 29. ,248 1, .00 33 .62 0
ATOM 6038 0 HOH w 193 30 .257 27 .659 31. ,979 1, .00 37 .42 o
ATOM 6041 0 HOH w 194 16, .442 32, .431 6. .611 1. .00 24 .66 o
ATOM 6044 0 HOH w 195 34, .073 12, .318 2. .203 1. .00 35 .07 o
ATOM 6047 0 HOH w 196 4 .395 17 .038 28. .248 1, .00 26 .36 o
ATOM 6050 0 HOH w 197 33 .318 3, .825 31. .828 1, .00 23 .35 o
ATOM 6053 0 HOH w 198 18, .983 33 .274 37. .816 1, .00 19 .94 o
ATOM 6056 0 HOH w 199 13, .726 12, .394 40. .361 1. .00 26 .01 o
ATOM 6059 0 HOH w 200 12, .010 7, .108 2. .281 1, .00 28, .49 0
ATOM 6062 0 HOH w 201 17 .870 4 .803 31. .837 1, .00 28 .97 0
ATOM 6065 0 HOH w 202 27 .323 19 .119 43, .497 1, .00 31 .89 o
ATOM 6068 0 HOH w 203 24. .085 33 .024' 8. .818 1, .00 27 .59 o
ATOM 6071 0 HOH w 204 19, .302 -7 .180 37. .095 1, .00 25 .01 o
ATOM 6074 0 HOH w 205 34, .921 3, .097 15. .744 1, .00 35 .90 0
ATOM 6077 0 HOH w 206 22 .046 36 .365 19, .883 1, .00 33 .71 0
ATOM 6080 0 HOH w 207 4 .178 27 .080 39, .663 1, .00 28 .68 0
ATOM 6083 0 HOH w 208 21 .450 25 .719 2, .335 1, .00 29 .50 o
ATOM 6086 0 HOH w 209 7, .625 37 .543 22. .457 1, .00 27 .39 o
ATOM 6089 0 HOH w 210 27 .905 0 .363 30, .805 1 .00 27 .13 o
ATOM 6092 0 HOH w 211 29 .963 26 .817 34, .574 1, .00 28 .33 0
ATOM 6095 0 HOH w 212 37 .812 29 .413 15, .021 1, .00 35 .13 o
ATOM 6098 0 HOH w 213 31, .600 5 .299 33. .905 1, .00 30 .36 o
ATOM 6101 0 HOH w 214 0, .934 2, .893 30. .953 1, .00 26 .27 o
ATOM 6104 0 HOH w 215 15 .151 -19 .091 27. .503 1 .00 36 .44 o
ATOM 6107 0 HOH w 216 31 .891 29 .580 32, .140 1, .00 23 .31 0
ATOM 6110 0 HOH w 217 13 .828 -7 .788 38, .638 1, .00 33 .17 o
ATOM 6113 0 HOH w 218 37, .026 8 .221 8. .178 1, .00 43 .15 o
ATOM 6116 0 HOH w 219 12 .026 -5 .374 16, ,938 1 .00 29 .86 o
ATOM 6119 0 HOH w 220 -1 .767 -3 .163 19, .781 1, .00 23 .61 0
ATOM 6122 0 HOH w 221 5, .748 3 .990 37, .161 1, .00 34 .03 o
ATOM 6125 0 HOH w 222 15 .126 10 .026 2. ,394 1, .00 29 .52 o
ATOM 6128 0 HOH w 223 28, .930 25, .732 2. .063 1, .00 32 .92 o
ATOM 6131 0 HOH w 224 17 .834 38 .165 18, ,660 1 .00 32 .25 o
ATOM 6134 0 HOH w 225 15 .576 -9 .633 19, .956 1. .00 29 .35 0
ATOM 6137 0 HOH w 226 21 .532 33 .500 36, .344 1, .00 30 .91 0
ATOM 6140 0 HOH w 227 37 .166 25 .308 14, .969 1, .00 30 .80 o ATOM 6143 O HOH W 228 4,.201 13.978 20..632 1..00 29,.14 o
ATOM 6146 O HOH W 229 -8. .921 0 .073 20. .951 1. .00 29, .35 o
ATOM 6149 O HOH W 230 30 .930 14 .280 0, ,673 1, .00 42 .54 0
ATOM 6152 O HOH W 231 0, .993 -10 .363 23. ,294 1, ,00 35, .64 o
ATOM 6155 O HOH W 232 19 .283 -9 .456 35, .875 1, .00 21 .72 o
ATOM 6158 O HOH W 233 29 .715 33 .139 9, .438 1, .00 28 .66 o
ATOM 6161 O HOH W 234 2 .904 -7 .322 21, .953 1, .00 29 .37 0
ATOM 6164 O HOH W 235 -0. .395 23 .877 34, ,029 1, .00 37, .81 0
ATOM 6167 O HOH W 236 15 .054 -3 .907 38, .561 1 .00 26 .91 0
ATOM 6170 O HOH W 237 25 .729 34 .682 11, .457 1, .00 29 .23 o
ATOM 6173 O HOH W 238 9 .385 33 .323 24, .057 1, .00 36 .55 o
ATOM 6176 O HOH W 239 24 .093 -5 .021 21, ,077 1, .00 26, .10 o
ATOM 6179 O HOH W 240 34, .767 17 .185 37. .911 1, ,00 29, .03 0
ATOM 6182 O HOH W 241 18 .069 25 .299 24, .326 1, .00 23 .95 o
ATOM 6185 O HOH W 242 25 .539 23 .840 -0, .133 1, .00 27 .32 0
ATOM 6188 O HOH W 243 -8, .581 -0 .882 24. ,380 1, ,00 32. .46 0
ATOM 6191 O HOH W 244 37, .140 34 .955 20. .025 1. ,00 39, .20 0
ATOM 6194 O HOH W 245 25, .828 -6, .464 17. .951 1. .00 35, .90 o
ATOM 6197 O HOH W 246 20 .526 5 .042 2. .568 1, .00 23 .17 o
ATOM 6200 O HOH W 247 16, .909 37 .789 30. ,355 1. .00 24, .49 0
ATOM 6203 O HOH W 248 4, .170 -13 .753 24. .179 1. .00 34, .75 o
ATOM 6206 O HOH W 249 4, .757 29 .554 36. .890 1. .00 27, .07 o
ATOM 6209 O HOH W 250 1 .985 25 .383 44, .611 1, ,00 36 .67 o
ATOM 6212 O HOH W 251 21 .002 34 .942 26, .743 1. .00 24 .40 0
ATOM 6215 O HOH W 252 35, .187 37 .614 16. .171 1, ,00 41, .70 0
ATOM 6218 O HOH w 253 9, .429 35 .849 24, .299 1. .00 29, .47 o
ATOM 6221 0 HOH w 254 22 .360 -8 .508 14. .886 1, .00 39 .42 o
ATOM 6224 0 HOH w 255 27 .125 28 .229 2. .829 1. .00 36 .93 0
ATOM 6227 0 HOH w 256 7 .686 9 .225 33, .089 1, .00 35 .16 o
ATOM 6230 0 HOH w 257 4 .744 8 .641 8, .479 1. .00 31 .36 o
ATOM 6233 0 HOH w 258 43. .322 15 .064 19, .229 1, .00 34 .65 0
ATOM 6236 0 HOH w 259 12 .158 34 .202 31, .572 1. .00 22 .62 0
ATOM 6239 0 HOH w 260 40 .415 22 .262 16, .091 1, .00 26 .05 o
ATOM 6242 0 HOH w 261 7 .689 33 .643 34, .608 1, .00 27 .52 o
ATOM 6245 0 HOH w 262 -2 .516 -11 .500 29. .608 1. .00 27 .56 0
ATOM 6248 0 HOH w 263 23, .197 30 .603 38. .577 1. .00 30, .81 o
ATOM 6251 0 HOH w 264 1 .669 -4 .135 18. .399 1, .00 34 .72 o
ATOM 6254 0 HOH w 265 31 .682 18 .313 2. .510 1. .00 27 .38 0
ATOM 6257 0 HOH w 266 21, .515 33 .588 40. .094 1. .00 30, .03 o
ATOM 6260 0 HOH w 267 16, .458 13 .271 -0. .901 1. .00 38, .54 o
ATOM 6263 0 HOH w 268 40, .177 32, .128 16. .843 1. .00 41, .26 o
ATOM 6266 0 HOH w 269 12 .143 -2 .734 15, .885 1. .00 27 .02 0
ATOM 6269 0 HOH w 270 27 .486 -3 .196 12. .318 1. .00 30 .88 0
ATOM 6272 0 HOH w 271 15, .668 6 .307 39. .384 1. .00 31, .07 o
ATOM 6275 0 HOH w 272 7, .819 6 .715 30. .569 1. .00 22, .48 o
ATOM 6278 0 HOH w 273 29 .983 17 .529 41. .917 1, .00 33 .57 o
ATOM 6281 0 HOH w 274 2 .674 6 .648 20. .688 1. .00 34, .77 o
ATOM 6284 0 HOH w 275 16, .983 2 .502 33. .738 1. .00 29, .85 o
ATOM 6287 0 HOH w 276 18, .800 36 .320 34. .162 1. .00 30, .58 o
ATOM 6290 0 HOH w 277 12 .363 24 .605 -1, .596 1, .00 34 .55 0
ATOM 6293 0 HOH w 278 14 .702 17 .110 -5, .593 1 .00 27 .29 0
ATOM 6296 0 HOH w 279 40 .591 12 .389 6, .204 1. .00 33 .96 o
ATOM 6299 0 HOH w 280 31 .608 16 .687 1, .057 1, .00 38 .88 0
ATOM 6302 0 HOH w 281 23 .897 -11 .952 20, .222 1, .00 34 .68 0
ATOM 6305 0 HOH w 282 11 .219 39 .478 21, .517 1, .00 33 .54 0
ATOM 6308 0 HOH w 283 2 .552 16 .703 24, .563 1, .00 35 .86 o
ATOM 6311 0 HOH w 284 27 .258 9 .495 42, .694 1, .00 30 .97 o
ATOM 6314 0 HOH w 285 5 .535 8 .881 16, .549 1, .00 28 .65 0
ATOM 6317 0 HOH w 286 2 .189 24 .192 36, .099 1, .00 31 .47 o
ATOM 6320 0 HOH w 287 19 .058 -1 .798 12, ,329 1 .00 34 .23 0
ATOM 6323 0 HOH w 288 10 .635 34 .933 33, ,408 1, .00 30 .86 o
ATOM 6326 0 HOH w 289 4 .333 -0 .576 36, .893 1, .00 44 .65 0
ATOM 6329 0 HOH w 290 25 .069 21 .389 -1, .195 1, .00 37 .85 0 ATOM 6332 O HOH W 291 28,.073 7,.826 39..103 1..00 28,.63 o
ATOM 6335 O HOH W 292 14, ,225 35. .260 33. .101 1. .00 43, .65 o
ATOM 6338 O HOH W 293 18, .965 -2. .916 15. .137 1. .00 38, .32 o
ATOM 6341 O HOH W 294 40, .370 19. .000 13. .716 1. ,00 33, .50 o
ATOM 6344 O HOH W 295 6, ,261 32, .033 33, .639 1. .00 30 .04 o
ATOM 6347 O HOH W 296 13, .696 37, .943 17, .160 1. ,00 36, .07 0
ATOM 6350 O HOH W 297 2, ,518 21, .734 35. ,950 1. .00 40, ,41 o
ATOM 6353 O HOH W 298 31, .821 26. .389 38. .109 1. .00 33, .84 o
ATOM 6356 O HOH W 299 -1, .406 2, .015 20. .824 1. .00 32 .14 o
ATOM 6359 O HOH W 300 27, .841 5. .928 34. .623 1. ,00 31 .37 0
ATOM 6362 O HOH W 301 33, .128 25. .346 29. ,949 1. .00 26 .11 o
ATOM 6365 O HOH W 302 16, .952 35, .715 12. ,956 1. .00 35, .08 o
ATOM 6368 O HOH W 303 19, .607 39. .276 19. .928 1. .00 43, .31 o
ATOM 6371 O HOH W 304 31, .667 24, .286 33. .803 1, ,00 31 .06 o
ATOM 6374 O HOH W 305 9 .682 34 .631 35 .657 1, .00 36 .70 o
ATOM 6377 O HOH W 306 24 .913 37 .958 16, .991 1, .00 33 .31 o
ATOM 6380 O HOH W 307 27 .526 -1 .443 8, .577 1, .00 31 .08 o
ATOM 6383 O HOH W 308 34, .923 14, .237 35, .665 1, .00 33 .29 o
ATOM 6386 O HOH W 309 23 .480 3 .819 33 .037 1. .00 28 .70 0
ATOM 6389 O HOH W 310 39 .917 30 .468 13, .351 1, .00 43 .99 o
ATOM 6392 O HOH W 311 20 .005 30 .742 39, .713 1, .00 31 .34 o
ATOM 6395 O HOH W 312 1, .762 19, .900 10, .728 1, .00 33 .53 o
ATOM 6398 O HOH W 313 21 .282 35 .887 11. .600 1, .00 34 .66 o
ATOM 6401 O HOH W 314 22 .512 -2 .583 9, .740 1, .00 30 .42 o
ATOM 6404 0 HOH w 315 19 .079 3 .993 34, .105 1, .00 40 .61 o
ATOM 6407 0 HOH w 316 2 .068 20, .663 16, .198 1, ,00 29 .05 0
ATOM 6410 0 HOH w 317 2, .691 6, .046 36, ,126 1. .00 41 .50 o
ATOM 6413 0 HOH w 318 34 .645 5 .648 8, .339 1, ,00 37 .23 o
ATOM 6416 0 HOH w 319 23 .607 9 .100 43, .132 1, .00 28 .98 o
ATOM 6419 0 HOH w 320 32 .041 25, .470 4, .056 1, ,00 37 .21 0
ATOM 6422 0 HOH w 321 20, .362 8, .689 26. ,048 1. ,00 25 .83 o
ATOM 6425 0 HOH w 322 11 .708 6 .948 24. .652 1, .00 11 .83 o
ATOM 6428 0 HOH w 323 27 .069 1 .261 5, .299 1. .00 22 .09 o
ATOM 6431 0 HOH w 324 23 .654 25 .543 41, .612 1, ,00 22 .43 o
ATOM 6434 0 HOH w 325 23, .776 33, .886 17, ,441 1, ,00 26 .34 o
ATOM 6437 0 HOH w 326 34, .498 18, .045 27. ,924 1. .00 26 .83 o
ATOM 6440 0 HOH w 327 34 .129 9 .041 28, .001 1, .00 29 .40 o
ATOM 6443 0 HOH w 328 22 .398 -4 .833 18, .306 1, .00 31 .97 0
ATOM 6446 0 HOH w 329 32 .650 36, .061 13, ,569 1, ,00 32 .16 o
ATOM 6449 0 HOH w 330 18, .875 6, .699 27, .476 1. .00 31 .00 o
ATOM 6452 0 HOH w 331 43, .627 18, .936 5, .210 1. .00 33, .15 o
ATOM 6455 0 HOH w 332 13 .390 23 .630 -3, .991 1, .00 34 .31 0
ATOM 6458 0 HOH w 333 -0 .102 6 .438 34, .964 1, ,00 48 .55 0
ATOM 6461 0 HOH w 334 -0, .118 6, .687 37, ,269 1, ,00 35 .34 o
ATOM 6464 0 HOH w 335 37, .771 7, .069 11, ,352 1. ,00 32 .60 o
ATOM 6467 0 HOH w 336 31 .257 24 .829 41, .652 1, .00 34 .05 0
ATOM 6470 0 HOH w 337 10 .129 21 .939 40, .735 1, ,00 34 .24 0
ATOM 6473 0 HOH w 338 6, .286 34, .026 36, .723 1. .00 34 .35 0
ATOM 6476 0 HOH w 339 10, .691 33, .573 28. .674 1. .00 35, .22 o
ATOM 6479 0 HOH w 340 12, .399 2, ,285 25. .575 1. .00 35, .31 o
ATOM 6482 0 HOH w 341 34 .307 9 .058 2, .642 1. ,00 42 .37 o
ATOM 6485 0 HOH w 342 15 .597 2, .822 28. .675 1. ,00 34 .26 o
ATOM 6488 0 HOH w 343 11, .007 37, .789 24. .517 1. .00 39 .01 o
ATOM 6491 0 HOH w 344 6, .436 -13, .362 22. .903 1. .00 41, .47 o
ATOM 6494 0 HOH w 345 19 .680 17 .126 45, .857 1. .00 37 .81 0
ATOM 6497 0 HOH w 346 10 .533 36, .113 27, .412 1. ,00 34 .50 o
ATOM 6500 0 HOH w 347 41, .809 19, .887 16, ,869 1. .00 37 .61 o
ATOM 6503 0 HOH w 348 21, .611 15, .053 -0. ,998 1. .00 45, .55 o
ATOM 6506 0 HOH w 349 22, .337 -9, ,031 17. ,861 1. .00 49, .31 0
ATOM 6509 0 HOH w 350 9 .303 25 .984 -1, .560 1, .00 39 .69 0
ATOM 6512 0 HOH w 351 13 .153 3 .923 27 .389 1 .00 35 .74 o
ATOM 6515 0 HOH w 352 20 .365 4 .572 37 .533 1, .00 43 .93 o
ATOM 6518 0 HOH w 353 -2 .246 27 .322 10, .724 1, .00 42 .74 o ATOM 6521 O HOH W 354 9,.435 33.000 4..908 1..00 38..59 o
ATOM 6524 O HOH W 355 15, .473 17, .843 44, .371 1, .00 40. .00 0
ATOM 6527 O HOH W 356 13. .009 31, .258 32, .197 1. .00 21. .10 o
ATOM 6530 O HOH W 357 36. .838 8, .242 3. ,607 1. .00 35. ,04 o
ATOM 6533 O HOH W 358 30, .674 -0 .077 10. .899 1. .00 34. .69 o
ATOM 6536 O HOH W 359 31, .146 37, .498 19. .980 1. .00 36. .95 0
ATOM 6539 O HOH W 360 16, .880 -0, .664 8. .461 1. .00 39. .07 0
ATOM 6542 O HOH W 361 40, .707 6, .845 20. .023 1. .00 32, .40 o
ATOM 6545 O HOH W 362 19, .502 25 .524 0, .606 1. .00 41 .49 o
ATOM 6548 O HOH W 363 27, .574 22 .139 44. .653 1. .00 52 .17 o
ATOM 6551 O HOH W 364 11, .308 11 .015 41. .537 1. .00 41, .08 o
ATOM 6554 O HOH W 365 9, .385 -15, .147 33. .423 1. .00 33, .24 o
ATOM 6557 O HOH W 366 8. .340 36, .413 6. .953 1. .00 46, .38 o
ATOM 6560 O HOH W 367 1 .749 16 .224 29. .518 1. .00 42 .28 o
ATOM 6563 O HOH W 368 21. .762 29 .936 41, .718 1. .00 39 .52 0
ATOM 6566 O HOH W 369 5, .916 6 .084 8. .101 1. .00 42, .08 o
ATOM 6569 O HOH W 370 22, .021 34 .841 24. .376 1. .00 41, .54 o
ATOM 6572 O HOH W 371 21. .487 -18. .393 21. .594 1. .00 38, .90 o
ATOM 6575 O HOH W 372 39 .073 5 .237 9, .752 1. .00 40 .75 o
ATOM 6578 O HOH W 373 23. .013 -4 .111 6, .967 1. .00 39 .88 o
ATOM 6581 O HOH W 374 14. .536 36 .281 12. .246 1. .00 37 .61 0
ATOM 6584 O HOH W 375 29, .859 34 .515 20. .996 1. .00 28, .10 o
ATOM 6587 O HOH W 376 28 .570 38 .564 14, .823 1. .00 51 .95 o
ATOM 6590 O HOH W 377 33 .330 21 .383 4, .315 1. .00 36 .55 o
ATOM 6593 O HOH w 378 44. .550 14 .418 11, ,401 1. .00 45 .79 o
ATOM 6596 O HOH w 379 20, .051 17 .411 -1. ,528 1. .00 37 .86 o
ATOM 6599 0 HOH w 380 0, .588 25 .706 15. .094 1. .00 42, .46 o
ATOM 6602 0 HOH w 381 4 .339 -3 .887 18, .462 1. .00 42 .68 0
ATOM 6605 0 HOH w 382 15 .219 38 .779 25, ,987 1. .00 45 .72 o
ATOM 6608 0 HOH w 383 26, .263 -1 .807 0. .657 1. .00 35, .43 0
ATOM 6611 0 HOH w 384 43, .222 8. .889 13. .058 1. .00 39, .12 o
ATOM 6614 0 HOH w 385 3 .804 15 .072 37. .146 1, .00 35 .83 o
ATOM 6617 0 HOH w 386 13 .685 22 .152 44. .161 1. .00 46 .34 o
ATOM 6620 0 HOH w 387 39 .745 6 .714 14. ,451 1. .00 43 .87 0
ATOM 6623 0 HOH w 388 4, .160 10 .246 14. ,729 1. .00 40, .70 o
ATOM 6626 0 HOH w 389 8, .951 3. .749 5. ,381 1. ,00 35. .43 o
ATOM 6629 0 HOH w 390 12 .861 -18 .451 24, .960 1. .00 37 .73 o
ATOM 6632 0 HOH w 391 2, .829 15 .459 20, .671 1. .00 39 .80 o
ATOM 6635 0 HOH w 392 13. .542 1 .619 37, .956 1. .00 42, .36 o
ATOM 6638 0 HOH w 393 16, .727 31 .361 39, .312 1. .00 38, .23 o
ATOM 6641 0 HOH w 394 36, .063 3 .510 12. .324 1. ,00 41, .15 o
ATOM 6644 0 HOH w 395 5 .800 1 .387 38. .322 1. .00 38 .68 0
ATOM 6647 0 HOH w 396 12 .445 36 .715 28, .596 1. .00 36 .10 o
ATOM 6650 0 HOH w 397 2, .782 12 .760 27, .641 1. .00 47 .29 o
ATOM 6653 0 HOH w 398 -1 .700 -3 .625 37. .395 1. .00 36, .77 o
ATOM 6656 0 HOH w 399 41 .093 10 .367 21. .318 1, .00 46 .59 0
ATOM 6659 0 HOH w 400 21 .734 -5 .069 9. .760 1, .00 48 .11 o
ATOM 6662 0 HOH w 401 14 .290 3 .253 24. .475 1. .00 36 .49 o
ATOM 6665 0 HOH w 402 22 .729 2 .974 -0, ,954 1. .00 40 .03 o
ATOM 6668 0 HOH w 403 4 .597 11 .469 0. ,380 1. .00 42, .88 o
ATOM 6671 0 HOH w 404 17 .898 4 .552 -0, ,927 1, .00 39 .85 0

Claims

1. A TY145 like subtilase which is at least 63% homologous to the sequence of SEQ ID NO:1 , comprising the overall subtilisin fold and the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, wherein the Strong ion-binding site of the BPN' like subtilases is not present, and with the exception of the TY145 subtilase, the S39 subtilase from TA39, the S41 subtilase from TA41 , and sphericase from B. sphaericus.
2. The subtilase of claim 1 , wherein the positions of said three ion-binding sites in the three- dimensional structure of the subtilase is defined by the distance to the c-alpha atoms of the three active site amino acid residues of the subtilases, that is Ser, His and Asp, and the c- alpha atom of the amino acid residue next to the active site Ser residue (next to Ser), wherein said distances between: a) the Weak ion-binding site and i) Asp c-alpha atom is 17.50-19.50A, ii) His c-alpha atom is 21-23A, iii) Ser c-alpha atom is 13.80-15.80A, iv) next to Ser c-alpha atom is 15.80- 17.80A, b) the Far ion-binding site and i) Asp c-alpha atom is 28.70-30.70A, ii) His c-alpha atom is 28-30A, iii) Ser c-alpha atom is 20-22A, iv) next to Ser c-alpha atom is 19.50-21.50A, c) the Near ion-binding site and i) Asp c-alpha atom is 27-29A, ii) His c-alpha atom is 29.50- 31.50A, iii) Ser c-alpha atom is 21.40-23.40A, iv) next to Ser c-alpha atom is 22.50- 24.50A,
3. A subtilase according to claim 2 wherein the positions of the three ion-binding sites are defined by the distance to the c-alpha atoms of amino acid residues D35, H72, S251 and M252 of SEQ ID NO:1 or by the distances to the c-alpha atoms of equivalent amino acid residues in another subtilase of the invention in accordance with claim 1 , wherein the distance between a) the Weak ion-binding site and i) D35 c-alpha atom is 18.55A, ii) H72 c-alpha atom is 21.98A, iii) S251 c-alpha atom is 14.71A, iv) M252 c-alpha atom is 16.75A, b) the Far ion-binding site and i) D35 c-alpha atom is 29.68A, ii) H72 c-alpha atom is 29.10A, iii) S251 c-alpha atom is 20.96A, iv) M252 c-alpha atom is 20.35A, c) the Near ion-binding site and i) D35 c-alpha atom is 28.04A, ii) H72 c-alpha atom is 30.43A, iii) S251 c-alpha atom is 22.28A, iv) M252 c-alpha atom is 23.58A, and wherein the variation on the above mentioned distances are ±O.δA, preferably ±0.7A, more preferably ±0.6A, more preferably ±O.δA, more preferably ±0.4A, or most preferably +0.3A.
4. A method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 like subtilase on the three-dimensional structure of a TY145 subtilase to produce a three-dimensional structure of the parent TY145 like subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent TY145 subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions corresponding to said structural part and e) expressing the modified nucleic acid sequence in a host cell to produce the variant TY145 subtilase.
5. A method according to claim 4, wherein the TY145 subtilase on which the parent TY145 subtilase is modelled in step a) is at least 63% homologous to SEQ ID NO:1 , preferably at least 65% homologous, more preferably at least 70%, more preferably at least 74%, more preferably at least 80%, more preferably at least 83%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or even more preferably at least 99% homologous to the sequence of SEQ ID NO:1.
6. A method according to claims 4 or 5, wherein the TY145 subtilase on which the parent TY145 subtilase is modelled in step a) is defined in accordance with claim 3.
7. A method of producing a variant of a parent Subtilisin family subtilase, the variant having at least one altered property as compared to the parent Subtilisin family subtilase, the method comprising: a) modelling the parent Subtilisin family subtilase on the three-dimensional structure of a Subtilisin family subtilase to produce a three-dimensional structure of the parent Subtilisin family subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a TY145 like subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent Subtilisin family subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent Subtilisin family subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions corresponding to said structural part and e) expressing the modified nucleic acid sequence in a host cell to produce the variant Subtil- isin family subtilase.
8. A method according to claim 7, wherein the Subtilisin family subtilase on which the parent Subtilisin family subtilase is modelled in step a) is at least 61% homologous to SEQ ID NO:5, preferably at least 63% homologous, preferably at least 65% homologous, more preferably at least 70%, more preferably at least 74%, more preferably at least 80%, more preferably at least 83%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or even more preferably at least 99% homologous to the sequence of SEQ ID NO:5.
9. A method according to any of claims 7 and 8, wherein the TY145 subtilase of step b) is defined in accordance with claim 3.
10. A method according to any of claims 7-9, wherein the TY145 subtilase in step b) is at least 63% homologous with the sequence of SEQ ID NO:1, preferably at least 65% homologous, more preferably at least 70%, more preferably at least 74%, more preferably at least 80%, more preferably at least 83%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or even more preferably at least 99% homologous to the sequence of SEQ ID NO:1.
11. A method of producing a variant of a parent TY145 like subtilase, the variant having at least one altered property as compared to the parent TY145 like subtilase, the method comprising: a) modelling the parent TY145 like subtilase on the three-dimensional structure of a TY145 like subtilase to produce a three-dimensional structure of the parent TY145 like subtilase; b) comparing the three-dimensional structure obtained in step a) to the three-dimensional structure of a Subtilisin family subtilase; c) identifying on the basis of the comparison in step b) at least one structural part of the parent TY145 like subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent TY145 like subtilase to produce a nucleic acid sequence encoding deletion or substitution of one or more amino acids at a position corresponding to said structural part, or an insertion of one or more amino acid residues in positions corresponding to said structural part and e) expressing the modified nucleic acid sequence in a host cell to produce the variant TY145 like subtilase.
12. A method according to claim 11 , wherein the Subtilisin family subtilase of step b) is at least 61% homologous to SEQ ID NO:5, preferably at least 63% homologous, preferably at least 65% homologous, more preferably at least 70%, more preferably at least 74%, more preferably at least 80%, more preferably at least 83%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or even more preferably at least 99% homologous to the sequence of SEQ ID NO:5.
13. A method according to any of claims 11 and 12, wherein the parent TY145 like subtilase is defined in accordance with claim 3.
14. A method according to any of claims 11-13, wherein the parent TY145 like subtilase is at least 63% homologous with the sequence of SEQ ID NO:1, preferably at least 65% homologous, more preferably at least 70%, more preferably at least 74%, more preferably at least 80%, more preferably at least 83%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or even more preferably at least 99% homologous to the sequence of SEQ ID NO:1.
15. A variant subtilase comprising an alteration in one or more positions located at a distance of not more than 10A to one of the ion-binding sites of TY145, wherein the positions, as specified in SEQ ID NO:1 , located at a distance of not more than 10A to: a) the Weak ion-binding site are: 154, 155, 158, 164, 165, 166, 167, 168, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 211 , 220, 221 , 222, 223, 224, 225, 226, 227, 228, 277, 281 and 305, b) the Near ion-binding site are: 185, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 277, 281 , 299, 300, 301 , 304, 305, c) the Far ion-binding site are: 193, 198, 199, 201 , 202, 204, 216, 217, 219, 226, 227, 228, 229, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 and 307.
16. A subtilase variant according to claim 15 wherein the alterations are one or more of the substitutions I220S,T,D,E; T215S,D,E; G298A,S,T,D,E; G296A,S,T,D,E; V185T,D,E; I221N,D,T,E.
17. A TY145 like subtilase variant comprising the introduction of a ion-binding site corre- sponding to the Strong ion-binding site of the Subtilisin family subtilases, wherein said variant has a deletion of the region H83-G90, or at least one deletion of one amino acid residue in the region H83-G90, of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion of the sequence LNNSIG (SEQ ID NO: 48) between residues A82 and V91.
18. A TY145 like subtilase variant in which one or more ion-binding sites have been removed, wherein said variant comprises one or both of the alterations a) deletion of the region K290-D300, or at least one deletion of one amino acid residue in the region K290-D300 of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion between I289 and Y301 of the sequence GDS (SEQ
ID NO: 49) or DST (SEQ ID NO: 50), and preferably further comprising the substitution S303Y, b) deletion of the region N212-R224, or at least one deletion of one amino acid residue in the region N212-R224 of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion of a proline residue or an alanine residue between
G211 and D225.
19. A TY145 like subtilase variant comprising one or more alterations in one or more of the positions contained in the following highly mobile regions: 84, 85, 86, 87 and 88,
108, 109, 110, 111, 112, 113, 114, 115, 116 and 117, 141 , 142, 143, 144, 145 and 146, 150, 151 and 152, 169, 170 and 171, 200 and 201 ,
211 , 212, 213, 214, 215, 216, 217, 218, 219 and 220, 242 and 243,
268, 269 and 270.
20. A TY145 like subtilase variant comprising one or more alterations in one or more of the positions contained in the following mobile regions: 1, 2, 3, 4, 5, 6 and 7,
17, 18, 19, 20, 21 , 22 and 23,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 and 69,
84, 85, 86, 87, 88, 89, 90, 91 and 92, 107, 108, 109 and 110,
239, 240, 241, 242 and 243
265 and 266, wherein said alterations preferably are introduced in one or both of the regions 57-69 and 84-
92.
21. A TY145 like subtilase variant comprising one or more disulfide bridges introduced by one or more of the following modifications: G26C+A95C; A167C+T254C; R203C+G292C; V228C+A284C, wherein the positions corresponds to the positions in SEQ ID NO:1
22. A TY145 like subtilase variant comprising the substitution D116H,K,R.
23. A TY145 like subtilase variant comprising an alteration in one or more of the positions 18, 115, 185, 269 and 293 of SEQ ID NO:1 , wherein the preferred alterations are Q18P, D115P, V185P, T269P and I293P.
24. A TY145 like subtilase variant comprising an alteration in one or more of the positions contained in the following regions:
16, 17, 18, 19, 20, 21 and 22,
40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73,
118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130 and 131 , 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157,158, 159, 160 and 161 ,
275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293 and 294, wherein such alterations preferably are made in one or both the regions 40-73 and 140-161 , preferably in the sub-regions 65-73 and 140-150.
25. A TY145 like subtilase variant comprising an alteration in one or more of the following positions: 35,36,70,72,106,109,110,111,112,113,114,117,139,140,141 ,142,143,144,145, 147,150,167,168,169,170,171 ,172,173,174,177,180,207,239,247,148,149,150,151 and 252 of SEQ ID NO:1.
26. A TY145 like subtilase variant comprising an alteration in one or more of the following positions: V31 , V38, T79, V80, L81 , V188, T254, wherein preferred variants comprise one or more of V31 I, T79S and V80A.
27. A TY145 like subtilase variant comprising an alteration of an Asn-Gly sequence by deletion or substitution of at least one of the Asn or Gly residues, preferably the Asn residue.
28. The variant of claim 27 comprising substitution of the Asn and/or Gly residue with an amino acid residue selected from the group consisting of A, Q, S, P, T and Y.
29. The variant of claim 28, wherein the substitution is performed in one or more of the following positions:
B. sphaericus: 198-199, 240-241 TY145: 87-88, 109-110, 199-200 TA41: 83-84, 198-199 TA39: 88-89, 198-199
30. A TY145 like subtilase variant comprising an alteration of a tyrosine residue by deletion or substitution, preferably to phenylalanine.
31. The variant of claim 30, wherein the substitution is performed in one or more of the following positions:
B. sphaericus: 14, 91 , 102, 112, 155, 157, 172, 179, 201 , 206, 211, 218, 235, 239, 243, 292, 300,
TY145: 15, 39, 92, 103, 113, 156, 158, 202, 219, 240, 244, 287, 301, 307, TA41: 15, 91, 102, 112, 155, 157, 179, 201 , 218, 235, 243, TA39: 15, 61, 91, 102, 112, 155, 157, 173, 179, 201, 211, 218, 235, 243, 267, 281, 284, 292, 293, 296
32. A TY145 like subtilase variant comprising an alteration of a methionine residue residue by deletion or substitution, preferably to a serine or alanine residue.
33. The variant of claim 32, wherein the substitution is performed in one or more of the following positions:
B. sphaericus: 138, 251, TY145: 139, 252, TA41 : 1, 138,251 , TA39: 1 , 138, 251,
34. A Subtilisin family subtilase variant in which the Strong ion-binding site has been re- moved, wherein said variant comprises deletion of the region L75-G80 (BPN' numbering), or at least one deletion of one amino acid residue in the region L75-G80 (BPN' numbering), or a corresponding region in another Subtilisin family subtilase, and subsequent insertion of one or more amino acid residues, preferably insertion of the sequence GGSNG (SEQ ID NO: 51) of positions 84-88 of TY145 (SEQ ID NO:1) between A74 and V81 , and preferably further cόm- prising one or both of the substitutions L80Y and Q2A,N.
35. A BPN' like subtilase variant comprising one or more of the alterations V28I, A, L; I35V,A,L; T71S; I72A,G,V; A73L.G; M175V.A; and T224S.A, wherein preferred variants of Savinase comprises one or more of the substitutions V28I, I35V, T71S, I72A, A73L, M175V and T224S (BPN' numbering), especially variants comprising the combinations V28I+I35V, V28I+T71S, V28I+I72A, V28I+A73L, V28I+M175V, I35V+T71S, I35V+I72A, I35V+A73L, I35V+A73L, I35V+M175V, T71S +I72A, T71S +A73L, T71S +A73L, T71S +M175V, I72A +A73L, I72A +A73L, I72A +M175V, A73L +M175V.
36. A BPN' like subtilase variant comprising one or more of the following alterations: a) deletion of residues PSPSATLEQAVN (SEQ ID NO: 23) (positions 129-140) in Savinase (BPN' numbering) and subsequent insertion of residues SAKDSLIASAVD (SEQ ID NO: 22) (positions 144-155) from TY145 between S128 and S141 in Savinase, b) deletion of residues SGNSGAGSISYPARYA (SEQ ID NO: 25) (positions 153-172) in Savinase (BPN' numbering) and subsequent insertion of residues
AGNSGSGSNTIGFPGGLV (SEQ ID NO: 24)(positions 168-185) from TY145 between A152 and N173 in Savinase, c) deletion of residues VNVQSTYPGSTYASLN (SEQ ID NO: 27) (positions 203-218) in Savinase (BPN' numbering) and subsequent insertion of residues ASVESTWYTGGYN- TIS (SEQ ID NO: 26) (positions 233-248) from TY145 between G202 and G219 in Savinase.
37. A BPN' like subtilase variant comprising one or more of the following alterations: a) deletion of residues LSLGSPS (SEQ ID NO: 29) (positions 124-130) in Savinase (BPN' numbering) and subsequent insertion of residues MSLGSSG (SEQ ID NO: 28) (positions 138-144) from TA39 subtilase between N123 and P131 in Savinase, b) deletion of residues LSLGSPSPSATL (SEQ ID NO: 31) (positions 124-135) in Savinase variant V104S (BPN' numbering) and subsequent insertion of residues MSLGSSGESSLI (SEQ ID NO: 30) (positions 138-149) from TA39 subtilase between N123 and E136 in Savinase variant V104S.
38. A BPN' like subtilase variant comprising one or more of the following alterations: a) deletion of residues VQAPAAHN (SEQ ID NO: 33) (positions 11-18) in Savinase (BPN' numbering) and subsequent insertion of residues NNSSITQT (SEQ ID NO: 32) (positions 16-23) from TA39 subtilase between R10 and R19 in Savinase, b) deletion of residues VPG*EPST (SEQ ID NO: 35) (positions 51-58) in Savinase (BPN' numbering) and subsequent insertion of residues TΛ GTTYTN (SEQ ID NO: 34 positions
56-63 from TA39 subtilase between F50 and Q59 in Savinase, c) deletion of residues GN (positions 61-62) in Savinase (BPN' numbering) and subsequent insertion of residues RQ (positions 69-70) from TA39 subtilase between D60 and G63 in Savinase. d) deletion of residues PSPSATL (SEQ ID NO: 37) (positions 129-135) in Savinase (BPN' numbering) and subsequent insertion of residues SGESSLI (SEQ ID NO: 36) (positions
143-149) from TA39 subtilase between S128 and E136 in Savinase. e) deletion of residues YPGSTYASL (SEQ ID NO: 39) (positions 209-217) in Savinase
(BPN' numbering) and subsequent insertion of residues WFDGGYATI (SEQ ID NO: 38) (positions 238-246) from TA39 subtilase between T208 and N218 in Savinase.
39. A BPN' like subtilase variant comprising one or more of the following alterations: a) deletion of residues VPG*EPST (SEQ ID NO: 41) (positions 51-58) in Savinase (BPN' numbering) and subsequent insertion of residues TΛ/GTNFTD (SEQ ID NO: 40) (posi- tions 56-63) from TA41 subtilase between F50 and Q59 in Savinase, b) deletion of residues ALNNSI (SEQ ID NO: 43) (positions 74-79) in Savinase (BPN1 numbering) and subsequent insertion of residues NGGTGS (SEQ ID NO: 42) (positions 83- 88) from TA41 subtilase between A73 and G80 in Savinase. c) deletion of residues ASGSGSV (SEQ ID NO: 45) (positions 98-104) in Savinase (BPN' numbering) and subsequent insertion of residues DDGSGYA (SEQ ID NO: 44) (positions 107-113) from TA41 subtilase between G97 and S105 in Savinase. d) deletion of residues KQKNPSW (SEQ ID NO: 47) (positions 235-241) in Savinase (BPN' numbering) and subsequent insertion of residues WAQSPAA (SEQ ID NO: 46) (positions 264-270) from TA41 subtilase between V234 and S242 in Savinase.
40. An isolated nucleic acid sequence comprising a nucleic acid sequence, which encodes for the subtilase or subtilase variant defined or produced in any of the preceding claims.
41. An isolated nucleic acid sequence according to claim 40, wherein the nucleic acid sequence is selected form the group consisting of: a) a nucleic acid sequence having at least 40% homology with the nucleic acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21 , and b) a nucleic acid sequence which hybridizes under low stringency conditions, preferably under medium stringency conditions, in particular under high stringency conditions, with c) a complementary strand of the nucleic acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21, or d) a subsequence of any of a), b) or c) of at least 100 nucleotides.
42. An isolated nucleic acid sequence according to claim 41 , wherein the nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21.
43. An isolated nucleic acid construct comprising a nucleic acid sequence as defined in any of claims 40-42, operably linked to one or more control sequences capable of directing the ex- pression of the polypeptide in a suitable expression host.
44. A recombinant host cell comprising the nucleic acid construct of claim 43.
45. A method for producing the subtilase or subtilase variant defined or produced in any of claims 1 to 39, the method comprising: a) cultivating the recombinant host cell of claim 41 under conditions conducive to the production of the subtilase variant; and b) recovering the variant.
46. A detergent composition comprising a subtilase or subtilase variant defined or produced in any of claims 1 to 39.
47. Use of a subtilase or subtilase variant defined or produced in any of claims 1 to 39 in cleaning or washing applications.
PCT/DK2004/000066 2003-01-30 2004-01-30 Subtilases WO2004067737A2 (en)

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