JP2002503538A - Kinetic controlled crystallization method and apparatus and crystals obtained by them - Google Patents

Abstract

(57) Abstract: A method and apparatus for kinetically controlling the crystallization of a molecule, the apparatus comprising: a crystallization chamber (s) for holding the molecule in a precipitant solution; 14) one or more precipitant solution reservoirs (16, 18), communication passages (17, 19), each of which connects the crystallization chamber (s) to each precipitant solution reservoir, and Transfer mechanism (20, 21, 22, 24, 26, 28), which is arranged to transfer the precipitant solution between each precipitant solution reservoir and the crystallization chamber (s), respectively. Is provided. These transfer mechanisms are interlocked to keep the volume of the precipitant solution in the crystallization chamber (s) constant. To adjust the concentration of the precipitant in the crystallization chamber (s), different concentrations of the precipitant solution are transferred into and out of the crystallization chamber (s) to achieve precise control of the crystallization process I do. This method and apparatus can be used effectively to reduce the crystallinity under reduced gravity conditions such as microgravity conditions in space, and under reduced or increased effective gravity conditions induced by strong magnetic fields. Grow.

Description

DETAILED DESCRIPTION OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a dynamically controlled crystallization system.
Controlled Crystallization System) (D
CCS), which accurately and kinetically processes the molecular crystallization (mol
Includes devices and methods that can be used to control the equatorial crystallization process.

The invention can further be used to remotely monitor and control the molecular crystallization process in real time.

[0003] The present invention can further be used to control the molecular crystallization process in a predetermined manner.

The present invention relates to the initiation, termination or reversal of a molecular crystallization process.
Enable.

[0005] The present invention makes it possible to reduce the size and number of movable parts of a molecular crystallization method and apparatus.

[0006] The present invention allows for the production of molecular crystals in a sealed system.

[0007] The present invention provides an earth's gravity field.
field) to produce molecular crystals under conditions of real and simulated gravity ranging from about 0 times to about 20 times the Earth's gravitational field and using the same equipment Can be used to produce crystals in the field.

The present invention allows for controlling crystallization parameters in a closed system without changing the pressure in the crystallization chamber.

The present invention can be used to optimize the time required to produce large and ordered molecular crystals.

[0010] The present invention can be used to optimize crystallization conditions while using less experimentation and substantially fewer reactants (eg, proteins).

The present invention is “kinetic control” as used herein, which means that (1) the crystallization process can be initiated at will, (2) the growth of molecular crystals Some conditions (eg, pH, conductivity, and temperature) that are important to the crystallization nucleation can be measured in the crystallization chamber, and And based on monitoring crystal growth,
(4) These conditions can be very precisely controlled at any time during the crystallization process, (5) The crystallization process can be adjusted or stopped if desired, (6) The process can be reversed if desired.

To achieve these objects, a first aspect of the present invention comprises: at least one crystallization chamber, which contains a solution, which contains reactant molecules. For retention, the solution being a crystallization chamber; at least one source reservoir, separated from the reagent solution (containing the precipitant at a first concentration) by a permeable membrane; A source reservoir comprising a reagent solution having a precipitant at a second concentration, the reagent solution being coupled to the reagent solution in the crystallization chamber via a communication passage; And at least one drain reservoir
a drain reservoir, which is coupled to the reagent solution in the crystallization chamber via a separate communication passage; and a transfer mechanism.
fer mechanisms, which respectively transfer the reagent solution between at least one source reagent solution reservoir and the crystallization chamber, and simultaneously transfer the reagent solution from the crystallization chamber to the at least one drain reservoir. A transfer mechanism configured to transfer. This transfer mechanism operates in line to simultaneously transfer an equal volume of the precipitate solution into and out of the crystallization chamber, thereby reducing the precipitation in the reagent solution within the crystallization chamber over time. The concentration of the agent can vary.
In this manner, the concentration of the precipitant in the reagent solution in the crystallization chamber can be kinetically adjusted to provide optimal crystallization conditions over time.

The present invention also provides that two or more pairs of source / drain reservoirs, each containing a different reagent solution, are each coupled to a reagent solution in the crystallization chamber via separate solution communication means. This means that for each pair of reagent reservoirs,
This is done by separate solution transfer means, transferring in series from the source reservoir to the crystallization chamber and from the crystallization chamber to the respective drain reservoirs. The source and drain reservoirs of this pair may be operated sequentially, ie, one pair at a time, and diffusion-mediated mixing of the reagent solution in the crystallization chamber during transfer.
With time left for diated mixing, the concentrations of reagents in the crystallization chamber and the precipitant in the reaction solution are also varied, in a controlled manner.

The present invention also provides at least one source reagent solution reservoir coupled to the first crystallization chamber reagent solution in series using two or more crystallization chambers in series via solution communication means. As well as with at least one drain reservoir coupled to the reagent solution of the last crystallization chamber in the series.
The serial transfer volume of the reagent solution from the source reservoir to the first crystallization chamber and from the last crystallization chamber to the drain reservoir is an equal volume of the next reagent solution from each crystallization chamber in the series. This results in a migration, whereby a gradient of precipitant in the reagent solution over a series of crystallization chambers is achieved.

[0015] The present invention also provides that two or more crystallization chambers are coupled to at least one source reagent solution reservoir via manifold solution communication means and also one or more drain reservoirs via solution communication means. Can be configured to be directly or indirectly coupled to, so that the same volume of reagent solution can be simultaneously transferred from the source reservoir to each of two or more different crystallization chambers.

By configuring the crystallization chamber of the present invention such that reactants in different crystallization chambers experience different ranges of precipitant concentrations and other parameters that affect crystallization (eg, multiple The present invention allows for controlled modification of the concentration of one or more precipitants during the process, thereby providing conditions for crystal growth, crystallization, This results in optimizing the process, terminating or reversing, and preserving the molecular crystals. Thus, the present invention allows the crystallization process to be easily controlled simply by operating the transfer mechanism.

Another important feature of the present invention is that the present invention has real-time, remotely controlled maneuverability, and that under normal earth gravity conditions, and where actual or simulated gravity fields It can be easily used in environments larger or smaller than the gravitational field.

The present invention also allows a complex set of variables in the process of crystal growth to be scrutinized, manipulated and controlled, so gravity is the only variable. Because of the simplicity and ease of operation, the present invention is conservative in terms of cost control for flying on the Space Shuttle and the International Space Station. Use of the present invention saves and uses both money and valuable astronaut time. The present invention can remain on a space station for many sets of experiments, while the crystallization chamber can return to earth with molecular crystals generated in space. New chambers, tubing and syringes can be delivered on the next flight pre-loaded. Furthermore, the present invention is easily miniaturized, which makes it even smaller than the embodiments shown in the embodiments disclosed herein, and thus the entire device is And can be even less expensive to move from space.

The present invention further provides that, under conditions where the effective attraction resulting from diamagnetism at high magnetic fields (ie, greater than 16 Tesla) ranges from 0 to 20 times the Earth's gravitational field, Methods for performing controlled crystallization of proteins and other molecules kinetically are provided.

Another important feature of the present invention is that the device is small and easily sealed to air. Sealing the system allows operation without adverse effects on fluid flow in the crystallization chamber and sieving characteristics of the dialysis membrane caused by air pressure. The seal prevents contamination of the solution in the system sample for further experiments.

A further feature of the present invention is that it has few moving parts, has low power requirements, and produces crystals in a reduced time.

DETAILED DESCRIPTION OF THE INVENTION While certain embodiments and preferred methods and materials of the present invention are described herein, the present invention is limited to the particular methodology, procedures, and materials described. Not done. Methods, materials, and devices similar or equivalent to those described herein,
While the invention can be used successfully in the practice and testing of the invention, the invention as described herein is capable of further modifications, and this application will generally apply to any of the various modifications, in accordance with the principles of the invention. It is intended to cover any uses, or indications, including any deviations from the present disclosure, which are known or conventional in the art to which this invention belongs, and are described herein. It is understood that essential features can be applied and are subject to the appended claims. All publications, patents, and patent applications mentioned herein are intended to describe and disclose the materials and methodologies reported in those publications, patents, and patent applications that may be used in connection with the present invention. And was incorporated as a reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein and in the appended claims, the singular forms "a,""an," and "the" refer to the parts having the same meaning. Unless otherwise indicated, plural references are included. For example, "a molecule to be crystallized" includes a plurality of this molecule.

The present invention includes an apparatus and method for molecular crystal production that allows for monitoring of crystal growth and kinetic control of crystallization conditions, whereby large, high quality crystals are relatively short. Generated in time. The method and apparatus of the present invention may comprise from about 10
It can be used to crystallize molecules ranging in size from small organic compounds of 30 atoms to large macromolecular complexes containing 10,000 atoms. The following description is
Often mention is made of using the methods and devices of the present invention to crystallize proteins, however, the methods and devices described herein may be used for molecules other than proteins, such as small organic molecules and intermediate size molecules. (Eg, oligonucleotides and oligopeptides) can also be used successfully to crystallize.

As used herein, the term “reactant” refers to a molecule that is to be crystallized.

As used herein, “precipitants” and “reagents” affect the solubility of the molecule to be crystallized and, depending on their concentration, promote or promote crystal growth in the crystallization process. Refers to compounds that can be inhibited. Examples of compounds that are precipitants are
Salts, polyethylene glycols, and small organic molecules (Durbin, S. & F
eher, G, Ann. Rev .. Phys. Chem. (1996) 47:17.
3).

As used herein, the term “reagent solution” refers to a solution containing one or more precipitants.

FIG. 1 shows a protein crystallization apparatus 10 of the present invention. Apparatus 10 includes a rigid base plate 12, a crystallization chamber 14 where crystallization occurs, a syringe 16, a syringe 18
, A lead screw 22, a yoke 24, and guide rods 26 and 28. The solution containing the reactant molecules to be crystallized is removably disposed within the crystallization chamber 14. The syringe 16 is provided by a tube 17 acting as a conduit
Coupled to the crystallization chamber 14. The syringe 18 is connected to the crystallization chamber 14 by a tube 19 serving as a conduit. Syringes 16 and 18
Serves as a reagent solution reservoir, as described in detail below. The plungers of the syringe 16 and the syringe 18 face each other and are coupled to a yoke 24 which serves as an interlock as described below. Further, the yoke 24 is arranged to slide on the guide rods 26 and 28. The lead screw 22 is coupled to the shaft of the motor 20 by an aligning coupling 21 and the yoke 24 is engaged by a threaded bushing. Syringe 1
6, the syringe 18, the guide rod 26 and the guide rod 28
The mounting plate 30 and the screw 31 allow the base plate 1
2 and fixedly connected. The mounting block 32 is fixed to the base plate 12 by screws 31 and has a bearing for allowing rotation of the lead screw 22, while the lead screw 22 is axially fixed to the base plate 12. Further, the crystallization chamber 14 and the stepping motor 20
Is fixed to the base plate 12 by a screw 31. The spring 29 is connected to the base plate 12 by a screw 31 at one end,
At the other end it is coupled to the yoke 24 to bias the yoke 24 in one direction against the threads of the lead screw 22, thereby minimizing backlash when the yoke 24 moves linearly as described below. Become The present invention operates successfully without the spring 29, and its use is optional.

The reactive molecules that can be crystallized according to the present invention include macromolecules (eg, proteins,
Nucleic acids in combination with proteins, lipids and proteins linked to glycolipids). If the reactive molecule is a membrane-bound protein, a detergent can be introduced into the crystallization chamber at an appropriate time, such as with a microsyringe, to separate the protein from the membrane. Reactive molecules also include small molecules, such as drug molecules, cofactors, amino acids, nucleotides, hormones and other signaling and regulatory molecules, and analogs described above. Intermediate size molecules that can be crystallized by using the present invention include, but are not limited to, peptides, protein fragments, and oligonucleotides. Large macromolecular complexes (eg, viruses) can also be crystallized using the present invention.

The crystallization chamber 14 holds the reactant molecules but includes a system for passing smaller molecules. A permeable membrane, porous diaphragm, or permeable sieve that cuts off the molecular weight for a given value may be used at this point. For example, the membrane may be a dialysis bag or a sheet comprising a dialysis membrane. In any case, the molecular weight cutoff should be less than the molecular weight of the reactant molecules in the crystallization chamber and should greatly reduce the motion of the reactant molecules outside the crystallization chamber. However, the membrane or sieve should not significantly impede the movement of smaller precipitant molecules (eg, ionic and non-ionic solutes). In one embodiment, a solution containing the reactant polymer to be crystallized is packed into dialysis sack 15. The dialysis sack 15 is placed directly in the crystallization chamber for holding the polymer. The reagent solution is then placed around the dialysis sack 15 to completely fill the crystallization chamber, as schematically shown in FIG. In a preferred embodiment, the crystallization chamber comprises a sieving membrane, such as a dialysis membrane, which divides the crystallization chamber into two compartments. One of the two compartments contains a reaction solution and the other contains a reagent solution. For example, a crystallization chamber can include a top cover that includes a depression. The cover itself may be recessed for this purpose, or the cover may comprise a button-shaped projection recessed for this purpose. Preferably, the cover comprises a recess which functions as a compartment for containing a solution containing reactant molecules for crystallization. The depression is then covered with the above-mentioned film. An elastic O-ring, gasket, or other retainer may be used to hold the membrane in place. A preferred embodiment of the crystallization chamber is shown in FIG. A transparent upper cover 49 having a columnar convex portion coincides with the inner cover 41. At this time, the central convex portion of the cover 49 coincides with the central hole, and the cover
A depression 42 is formed through the hollow columnar projection. The cover 49 is fixed in place with three screws. The depression 42 is filled with a reaction solution containing the molecules to be crystallized. The opening is covered with a dialysis membrane 43 held in place by an O-ring 44. Alternatively, the opening at the bottom of the perforated cylindrical protrusion of the inner cover 41 is closed by the dialysis membrane 43 held in place by the O-ring 44. Then, the storage tank 45 below the crystallization chamber 46 is filled with a desired reagent. An inner cover is then placed on the lower portion 46, the exposed surface of the dialysis membrane is in full contact with the reagent solution, and there are no bubbles in the crystallization chamber. The inner cover is then fixed in place with three screws. A reaction solution (eg, a solution containing the protein to be crystallized) is added to the well through a hole in the top of the inner cover. The depression has the upper surface of the dialysis membrane as its base. Then a transparent top cover 49 is placed on the inner cover. At this time, the center convex portion of the inner cover matches the center hole of the inner cover, and the transparent upper cover 49 is fixed in place with three screws. The cover seal 47 prevents the solution from leaking out of the chamber. When the two covers are constructed, the volume of the recess 42 formed is the diameter of the hole through the inner cover, the length of the hollow cylindrical protrusion extending from the inner cover, and the solid cylindrical protrusion extending from the upper cover. It is determined by the difference in length of the part. The volume of the depression 42 ranges from about 10 microliters to 5
It can range up to 00 microliters or more. The volume of the reagent solution reservoir 45 in the lower chamber can range from about 50 microliters to 10 milliliters or more. In a preferred embodiment, the volume of the depression 42 is about 50
The volume of the microliter and the reagent solution storage tank 45 is 1 milliliter. The present invention also includes other methods of retaining the reactant molecules in the crystallization chamber, which will be readily apparent to those skilled in the art.

The reagent solution is packed in a syringe 16 (optionally also in a syringe 18). Each reagent solution used in the crystallization chamber and the syringe may include one or more optional precipitants having ionic or chemical properties, which may crystallize proteins at appropriate concentrations. . The relative concentrations of the reagents in the crystallization chamber and the solution chamber can vary based on the crystallization conditions. Using the apparatus and method of the present invention, the concentration of the reagent solution in the crystallization chamber is raised or lowered to a desired concentration for crystallization.

A conduit for transporting the reagent solution between the paired reagent solution reservoir and the crystallization chamber is connected to a portion of the crystallization chamber. The crystallization chamber contains the reagent solution at any point sufficiently far away that the crystallization occurs before the reagent solution introduced into the crystallization chamber from the source reservoir is drawn into the conduit leading from the crystallization chamber to the drain reservoir. Mix well with the reagent solution in the chamber. For example, FIG. 1 shows tubing 17 and 1 from syringes 16 and 18, respectively, connected to the same side of crystallization chamber 14.
9 and FIG. 3 shows tubing from source and drain reagent solution reservoirs connected to opposite ends of the crystallization chamber. For further clarity, FIG. 3 is a cutaway view showing the threaded insertion of the inlet tube 48.

The start of the motor 20 causes the lead screw 22 to rotate, thereby causing the yoke 24 to slide linearly along the guide rods 26 and 28. York 2
4 moves the plunger of the syringe 16 in one direction and the plunger of the syringe 18 in the opposite direction over the same distance. Simultaneous opposing movements of the plungers in the two syringes cause one syringe to inject a small amount of reagent solution into the reagent reservoir of the crystallization chamber and the other syringe to remove the same volume of solution from the same reservoir. For example, the yoke 2 is moved in the direction of the arrow X shown in FIG.
When the motor 20 is started in the direction in which the cartridge 4 is moved, the reagent solution contained in the syringe 16 is moved into the crystallization chamber 14. Meanwhile, the reagent solution is simultaneously transferred from the crystallization chamber 14 into the syringe 18. If the concentration of the precipitant in the reagent solution (which results in the formation of crystals) is known, the respective concentrations of the precipitant in the reagent solution in the crystallization chamber 14, syringe 16 and syringe 18 are adjusted to an appropriate level before crystallization. It can be set to different values. The concentration of the precipitant of the reagent solution in the crystallization chamber 14 can then be controlled over time by the operation of the motor 20 in a desired manner over time. The optimum time to dynamically adjust the precipitant concentration in the crystallization chamber to a concentration that yields crystals of the desired size can then be empirically determined.

For example, for certain proteins, it has been found that crystallization is promoted at a salt concentration of about 0.15 M (mol / L) in the reagent solution. It is also desirable to approach this concentration very slowly, and it has been found that the speed of approach is important in obtaining the desired result. Thus, in a preferred embodiment,
Crystallization chamber 14 and syringe 18 may initially contain a reagent solution containing a sufficiently high concentration (eg, 1 M salt) of a precipitant such that crystallization does not occur. Syringe 16 is then used at a concentration lower than that allowing crystallization (eg, 0.1 M salt).
A reagent solution containing a precipitant may be initially included. With this configuration for initiating the crystallization process, the motor 20 can be activated to move the yoke 24 in the direction of arrow X in FIG. 1 so that a small amount of the low concentration reagent solution is crystallized from the syringe 16. Transfer into the chamber 14 and at the same time transfer the highly concentrated reagent solution from the crystallization chamber 14 into the syringe 18. It can be seen that this procedure reduces the concentration of the precipitant in the crystallization chamber 14. The motor 20 can be activated in a desired increment in a pre-programmed manner to change the concentration of the solution in the crystallization chamber 14 to a desired value over a selected time. In a preferred embodiment, the sensor is located in the crystallization chamber, in a syringe, or in a suitable location for detecting solution concentration, temperature, position, pressure or other variables, and using one or more monitoring systems. ,
Monitor crystal nucleation and crystal growth in the crystallization chamber. Measurements made by such sensors and monitoring systems during the crystallization process are used to control the crystallization process in a dynamic manner. Next, crystallization chamber 1
4 is at the optimal level (eg, 0.15M) for producing crystals.
Motor 20 can be stopped. And motor 2
Operating the 0 to move the yoke 22 in the opposite direction (ie, opposite the direction of the arrow X), solution may flow from the syringe 18 to the crystallization chamber 14 and from the crystallization chamber 14 to the syringe 16. Thereby, the concentration of the precipitant in the crystallization chamber 14 increases. In this manner, the concentration of the precipitant in the crystallization chamber 14 is fine-tuned,
The crystallization process can be raised or lowered to a value that can even stop or retreat. Thereby, precise dynamic control of the crystallization process is obtained.

[0034] Any suitable motor may be used. For example, motor 20 may be a stepper motor (e.g., model number STP42 ND 4BLVE-100 manufactured by EPSON AMERICAN). Similarly, as described below,
Using any type of controller (such as a digital or analog programmable controller) that facilitates certain programmable control of the crystallization process,
The motor 20 may be controlled (eg, MODERN TECHNOLOGY, IN
C. MTSD-V1 motor driver manufactured by the Company). For use in space, all components, including motors and controllers, may have to be certified or certified by appropriate regulatory bodies.

FIG. 2 is a schematic diagram of a controller 40 for controlling the motor 20, and thus is a crystallization process. The controller 40 includes a control circuit 50, an input connector 6
0, a programming terminal 70, a display 80, an output connector 90, and an input connector 100. The control circuit 50 includes a CPU (Central Processing Unit) 52
, I / O circuit 56, clock 54, and memory 53. The control circuit 50
It may be a conventional microprocessor-type device (such as a personal computer or an industrial programmable controller) or the control circuit 50 may be, for example, a dedicated solid-phase, hard-wired digital, analog or mechanical controller. . Clock 54 generates pulses with a regular period in a conventional manner and provides timing for the operation of other components. The memory 53 includes operation instructions for the CPU 52 and executes a crystallization process. The operating instructions may be in the form of programmed software in the case of a digital controller, hard-wired components in the case of an analog controller, or mechanical elements such as cams in the case of a mechanical controller. I / O circuit 56 provides an interface between CPU 52 and external components such as motor 20, programming terminal 70, display 80, and any sensors or actuators.

The input connector 60 has a motor voltage (+ M), a 5 volt D.C. C. Logic voltage (
+5), and three input connections for ground (G). The input connection of the connector 60 is connected to the I / O circuit 56. The output connector 90 includes a first winding (M1) of the motor 20, a second winding (M2) of the motor 20, and an output connection for ground (G). The output connection of the connector 90 is connected to the I / O circuit 56 and (a) (FIGS. 1 and 2).
Is connected to the stepping motor 20). The programming terminal 70 is connected to the CPU 52 via the I / O circuit 56,
2 to be input into the memory 53. Programming terminal 70 may comprise any input device such as a keyboard, touch screen, switch arrangement, push buttons, and the like. Once the operating instructions are stored in the memory 53, the programming terminal 70 can be disconnected from the I / O circuit 56 or the I / O circuit 56
May remain connected to the The display 80 is also connected to the CPU 52 via the I / O circuit 56, and functions to display operation parameters related to the operation of the controller 40 or their states. For example, the display 80 is a CRT,
It can be an LCD display, LED display or similar device. Of course, the display 80 is optional.

Once the operating instructions have been stored in memory 53 via conventional programming and storage techniques, the appropriate input voltage is provided to input connector 60 and output connector 90
Is connected to the stepping motor 20, and the controller 40 can control the motor 20 to operate in a predetermined manner with respect to time, thereby controlling the crystallization process in a desired manner. Naturally, if the initial concentrations of the precipitant in the reagent solution in the syringes 16 and 18 and the crystallization chamber 14 are known, and the volumes of the crystallization chamber 14, the syringe 16 and the syringe 18 are known, the memory 53 The stored operating instructions may be configured to control the crystallization process (i.e., the precipitation in the crystallization chamber 14 in a kinetic manner with respect to time to provide the desired crystallization conditions at any time). Control the concentration of the agent). Input connector 100 includes inputs I ₁ -I ₄ for connecting further inputs such as limit switches, sensors, etc. to CPU 52 via I / O circuit 56. This allows closed loop control to be achieved based on the state of the crystallization process or other variables. Further, these inputs are connected to switches and the like, and can activate, terminate, suspend, or change the operation of CPU 52. Of course, the operating instructions stored in memory 53 can be programmed with any desired operation and external inputs or outputs.

As mentioned above, one or more sensors may be connected to the crystallization chamber and fed back to enable closed loop control. For example, if the precipitant is a salt,
Sensors that measure the electrical resistance or other electrical properties of the salt solution in the crystallization chamber 14 may be used to indicate the concentration of the solution in the crystallization chamber. This is because the electrical properties of the salt solution change as the concentration changes. Additional sensors that measure the pH and temperature of the reagent solution in the crystallization solution may also be mounted on the crystallization chamber 14. Sensors suitable for use in the present invention for measuring ionic strength and pH are described, for example, in Microelectronics, Inc. , 40 Harvey
Rd. , Bedford, NH. Temperature sensors suitable for the specifications in the present invention are, for example, Bios from Colorado University Boulder, CO
Available from erve Space Technologies. FIG. 4 is a top view of a preferred embodiment of the crystallization chamber of the present invention, showing the configuration of the attachment of the pH reference probe 62, the conductive probe 63, the pH measurement probe 64, and the temperature probe 65. Also shown are an inlet tube 66, an outlet tube 67, and a screw 68 that secures the top of the crystallization chamber. FIG. 1 shows a single crystallization chamber 1
4 schematically shows an embodiment of the device of the invention comprising Ion intensity sensor 33, p
An H sensor 34 and a temperature sensor 35 are mounted on the crystallization chamber 14. Leads 36, 37 and 38 connect the sensor to ionic strength, pH, and temperature probes that come into contact with the reagent solution in crystallization chamber 14. Output line 56,
57 and 58 control ionic strength, pH, and temperature information (FIG. 2).
40) corresponding to one or more of the inputs I ₁ -I ₄ shown in FIG.

In a preferred embodiment of the present invention, crystal nucleation and crystal growth in the crystallization chamber are monitored in any manner known to those skilled in molecular crystal growth. For example, a crystallization chamber can have a transparent cover, and crystal nucleation and crystal growth observe the contents of a reagent reservoir or dialysis sac in the crystallization chamber with a suitable magnification device (eg, a microscope). Can be monitored by Observation and visual recording devices that reflect the interior of the crystallization chamber 14 are not shown, but such devices are commonly used to assess crystal nucleation and crystal growth and can be readily used by those skilled in the art with the present invention. . Such devices may include (but are not limited to) a camera for taking still pictures, a video recording device, a magnifying device, a tandem type magnifying device with a video recording device. Alternatively, early crystal growth is detected by directing the optical fiber leads to a reagent solution (and thereby directing light to the solution) and measuring light scattering (eg, Durbin and Feher, 1996, p. 184). Please refer to). As protein crystallization occurs from a reagent solution, the electrolytic properties of the reagent solution change. As a result, crystal nucleation can be detected by introducing a conductive wire into the reagent solution that functions as a plate for the capacitor. A change in the frequency of the RLC oscillator generated in this manner is an indication of crystal growth.

In a preferred embodiment, a syringe is used to contain the reagent solution, the plunger of the syringe is moved by the yoke, and the reagent solution is moved into and out of the crystallization chamber (s). In embodiments having more than one pair of source and drain syringes, each pair of syringes is connected to a different motor drive yoke, so that they can operate independently of each other. However, any configuration for delivering the reagent solution to and from the crystallization chamber can be used. For example, two or more reservoirs, each pair having a pump that is electrically or mechanically interlocked, may be used, or a single reservoir or syringe may be used to store some excess reagent solution. Emission into or to the atmosphere, or a valve configuration may be used. Any solution communication means may be used as a solution conduit for guiding the reagent solution between the reagent solution reservoir and the crystallization chamber. For example, plastic, glass, or metal tubing, or fluid transport channels in a chip (eg, silicon) are suitable for the present invention. Any mechanism, such as a software interlock or any other mechanical interlock programmed in the controller may be used to coordinate the transfer of the reagent solution to and from the crystallization chamber. Further, the reagent solution can be transferred to a test tube, beaker, or other suitable container, in which is placed a dialysis bag filled with a solution containing the molecules to be crystallized. A test tube, beaker, or other suitable container may then serve as the crystallization chamber, as described above, wherein the selected reagent solution is pumped into and out of the solution holding the dialysis bag. The composition of the solution around the dialysis bag, which is dispensed or injected and produces crystals in the crystallization chamber, is dynamically changed to a composition that produces crystals.

As mentioned above, the device of the present invention may include a set of multiple independently controlled syringes. A set of syringes is attached to the crystallization chamber. In such embodiments, a first set of syringes may be attached to the crystallization chamber and deliver a solution containing a precipitant suitable to promote nucleation and crystal growth. A second set of syringes can then be attached to the same crystallization chamber to deliver the appropriate salt-containing solution for stabilizing and storing the crystals over time. Once the appropriately sized crystals are generated by varying the concentration of the precipitant using the first set of syringes, the second syringe is used until the desired concentration stabilizes the salt solution in the crystallization chamber. From the set is contained in the reagent reservoir of the crystallization chamber via a very narrow hole access tube (<0.2 mm) or other suitable outlet, while an equal volume of fluid from this area is It can be pumped out of this area of the chamber simultaneously through a hole (<0.2 mm) or other suitable outlet.

The foregoing disclosure describes an embodiment of the present invention having a single crystallization chamber as shown in FIG. If the conditions to produce crystals of useful size and quality are known,
Using an apparatus having a single crystallization chamber, the conditions for efficiently changing the concentration of one or more precipitants (eg, salts) in the crystallization chamber in a controlled and controlled manner to provide crystals. Can give. Thereby, the time required to grow the crystal may be reduced or optimized. An apparatus having a single crystallization chamber is also useful for comparing crystal growth under different conditions (eg, different gravitational fields or different temperatures).

The crystallization conditions that result in a crystal in which a given protein or other molecule is well arranged include:
Little known or not optimized. In this case, conditions that promote good crystal growth may be found using embodiments of the apparatus of the present invention that include multiple crystallization chambers and that allow multiple solution parameter changes in different crystallization chambers. .

FIG. 5 schematically shows crystallization chambers in series connected by small diameter tubing. The first and last crystallization chambers are connected via tubing to a syringe containing a reagent solution of a selected concentration. The source and drain reservoirs referenced in FIG. 5 correspond to the syringes 16 and 18 of FIG. 1 and perform similar functions. In one embodiment, all crystallization chambers in series initially contain the same reactant and reagent solutions. Each trace of solution is injected from the source syringe into the first cell in series, and an equivalent volume of solution is transferred from each cell to the next adjacent series of cells.
It is moved to tubing leading "downstream" and from the last cell to the drain syringe. The reagent solution containing a high concentration of precipitant injected from each trace source syringe was mixed with the reagent solution in the first cell and diluted, and thus was transferred from the first cell to the second cell. The reagent solution has a lower concentration of precipitant than the trace reagent solution injected into the first cell. Dilution of the transferred reagent solution occurs in each subsequent cell in series in a similar manner. Thus, the rate of increase of the precipitant concentration is fastest in the crystallization chamber adjacent to the source reservoir and decreases continuously in each successive chamber in series. For complete control of the precipitant concentration in the cells, it is important that the reagent solution does not flow or diffuse between cells except during controlled transfer. Very small diameter tubing is used to connect the cells to prevent “cross flow”. Alternatively, a valve that is open only while liquid is being transferred between cells may be inserted into the tubing line between cells.

FIG. 6 schematically shows an embodiment of the present invention. The reagent solution in embodiments of the present invention is transferred from a source reservoir via a manifold to a plurality of crystallization cells, each of the plurality of crystallization cells may be changed in an initial reactant and / or reagent solution.
For this embodiment, the crystallization process can be monitored in a set of crystallization chambers with different initial conditions, as the concentration of one or more precipitants changes simultaneously in all of the cells in a regulated manner.

The present invention also includes configuring a crystallization chamber in a two-dimensional or three-dimensional matrix according to the techniques described above to analyze crystallization under a number of different kinetically controlled conditions. . FIG. 7 schematically shows a 2-D matrix of the crystallization chamber. Two different solutions (each solution containing one or more precipitants) are pumped in different directions through the rows and columns of the cell to create independently controlled concentration gradients of the precipitants. Each source and drain syringe operates in tandem, with different source / drain pairs being operated sequentially. Cross-flow between cells is prevented by connecting the cells using tubing with small holes,
If necessary, it is prevented by connecting a valve in the line between the cells that is open only when fluid is transferred.

In a similar manner, the effect of crystallization by changing the concentration of one or more additional precipitants in multiple crystallization chambers is to reduce the crystallization chambers of two or more matrices (eg, “stacks”). Recognized by joining, one or more gradients of precipitant concentration may be established to form a third dimension. Source and drain syringes that control the flow of additional reagent solutions into and out of the crystallization chamber of the first matrix and from the chamber of the last matrix are operated in tandem and operate in a manner similar to that described above for the 2-D matrix syringe. Activated sequentially for a set of syringes. Of course, there are countless modifications and combinations of the above-described configurations of the invention that can be used to analyze crystallization. Analysis of crystallization is, for example, by using multiple and independently controlled source syringes connected to the same or different cells in a given dimension, and by changing the initial conditions in the crystallization chamber. Such modifications of the methods and apparatus described herein are readily within the ability of those skilled in the art to produce molecular crystals and are included in the present invention.

In addition to controlling the concentration of one or more chemical precipitants to produce molecular crystals, the methods of the present invention can affect or promote crystal growth, or reduce the growth of crystal growth. Enables and includes control of physical parameters that can improve quality. For example, temperature is an important physical parameter affecting crystal growth, and the present invention involves the use of a device to control the temperature of the solution in the crystallization chamber. Examples of devices for controlling the crystallization temperature include Peltier coolers, temperature control vessels, and heating coils integrated into the system.

As mentioned above, another important physical parameter affecting crystal growth is gravity. Crystallization experiments conducted in outer space have shown that larger and more similar crystals
Microgravity (1 / 1000g to 1/1) by eliminating the influence of the earth's gravitational field
0,000 g). Example 2 below illustrates the successful operation of the present invention in producing protein crystals in the microgravity environment of space. The device of the present invention is rotated by a centrifuge in outer space,
Although the reactant molecules in the crystallization chamber are larger than the microgravity existing in space,
Can be exposed to gravity less than the normal earth's gravity. Alternatively, a centrifuge may be used in space or on Earth to expose the reactant molecules in the crystallization chamber to gravity greater than normal Earth gravity.

The advantage gained by growing crystals in reduced gravity in space is also that crystals can be grown in reduced effective gravitational fields, such as obtained from antiferromagnetic effects induced in extremely large magnetic fields. It can be obtained by growing. In a strong magnetic field, antiferromagnetism causes the electrons of the molecules in the magnetic field to undergo a repulsive or attractive force that depends on the strength of the magnet and the distance of the molecule from the center of the magnet. In a sufficiently strong magnetic field, there is a position with respect to the center of the magnetic field, where the material goes through zero effective gravity (g _eff = 0) and the material floats further (Beaugnnon et al., Nature (1991))
349: 470). When moved to a different position with respect to the center of the magnet, the material experiences an effective gravity in the range between zero and normal Earth gravity. At other positions with respect to the center of the magnet, the material will have between one and two
Subject to zero times the effective gravity. Also limited by the real-time, remote-controlled maneuverability, compactness, and ease of use that allow the devices and methods of the present invention to be used in the microgravity environment of outer space. To be used effectively within a defined environment. Extremely high magnetic fields are generated in a restricted environment to grow crystals in an effective gravitational field ranging from zero to about 20 times the gravity of the Earth's surface. A further advantage gained by growing crystals in magnetic fields that can produce strong antiferromagnetic forces is that molecules in such fields tend to align in the same direction, promoting the growth of ordered crystals. It is in.

In order to grow crystals under different effective gravity ranges, the vertical assembly of the crystallization chamber 71 supported by the support strip 72 may be provided with a high magnetic field (eg, 16T) hole 73 as shown in FIG. Is placed within. Each crystallization chamber consists of a reactant reservoir with a volume of about 1 ml. Reactant reservoir in communication with the reagent chamber through the dialysis membrane, as described above (volume of a few mm ^3). Thus, each crystallization chamber has a different g _eff value and a different magnetic field value. For a given field value, the crystallization chamber can be positioned at g _eff = 0 by adjusting the entire stack. It is important to note that the stack does not levitate. To be precise, for g _eff = 0 with little force over a distance less than 1 mm, there are microcrystals of the reactant in a particular cell. Each chamber in the stack _attempts a step change in g _eff for the position as shown in FIG. The chamber above the center of the magnet has g _eff <g and below the center of the magnet g _eff > g. The center chamber shows a control experiment when a magnetic field is present (where g _eff = g). The first crystallization chamber 71 shown in FIG. 8 is connected to a reagent solution source reservoir by an inlet tube 74. Small diameter tubing connects each crystallization chamber to the next in the series and the last chamber in the series is connected by a return tube 75 to the drain reservoir. The configuration of this crystallization chamber is the same as that shown in FIG. By controlling the flow of the reagent solution containing one or more precipitants through the series chamber, a concentration gradient of the precipitant can be established across the crystallization chambers in series. This measure allows for crystal growth in a multifactorial environment where both the precipitant concentration and the effective gravitational field change. Further, the total magnetic field can also vary. The total magnetic field can vary between about 16 and 33 Tesla. this is,
Shift the g _eff = (2 to 6) g and g _eff = 0 points further away from the center of the magnet. For fields smaller than 16T, the g _eff = 0 condition cannot be maintained, but smaller changes can be obtained for the condition 2 g <g _eff <g.

In one embodiment of the present invention, the magnet is a water-cooled resistance magnet, and the controller adjusts the temperature of the magnetic hole to a temperature between 10 ° C. and 40 ° C. once the steady state is reached. And may control additional physical parameters that affect crystal growth. In a different embodiment, the magnet is a superconducting magnet that is cooled and stored in a cryogenic vessel, the superconducting magnet being adapted to bring a magnetic field region of interest closer to a temperature selected by the controller (eg, room temperature). Similar to magnets used by magnetic resonance (NMR) spectrographers or clinical magnetic resonance imaging (MRI) specialists.

Example Carboxypeptidase-A (CPA) and Concanavalin-A (CON-
The crystals of A) are grown under normal earth gravity conditions (1 g) using the method and apparatus of the present invention.
). Using the present invention, crystals of ribonuclease S were grown both on the earth and in the microgravity environment of the space shuttle (Flight STS-95) onboard. The crystallization chamber in the equipment used for the experiment was 1 m
1 volume.

Example 1 Crystallization of CPA In an experiment using CPA, the protein was converted to 1.2 M LiCl pH 7.5.
The solution was placed in a dialysis bag at 50 microliters with a protein concentration of 12 mg / ml. The dialysis bag was then placed in the crystallization chamber and the remaining crystallization chamber was filled with a 1.2 M LiCl solution at pH 7.5. PH 7 in one of the syringes.
5 of a 0.1 M LiCl solution was injected into the crystallization chamber. Several trials were performed under these initial conditions. Different amounts of salt solution were transferred from the syringe to the crystallization chamber for each trial. In each trial, the amount of salt solution transferred from the crystallization chamber was equivalent to the amount of salt solution transferred to the crystallization chamber as described above. Each mixing time was less than one minute.

The results of the crystallization test on CPA are shown in Table I below.

[0056]

[Table 1] It can be shown that runs 1 and 2 did not reduce the concentration of the salt solution in the crystallization chamber enough to produce large crystals. However, in trials 3-5, a larger amount of the low concentration salt solution was transferred to the crystallization chamber, and thus the concentration of the salt solution was reduced to a range where proteins could precipitate out of solution and form large crystals. Was. The results of trials 3-5 were advantageously compared to control trials using a conventional dialysis method of crystallization. In a control trial, 50 μl dialysis bag was loaded with CPA (
(Concentration of 12 mg / ml in 1.2 M LiCl), pH 7.5. The dialysis bag was then hung in pH 7.5, 0.1 M LiCl (1 ml). Control trials resulted in large crystals, which could not be controlled kinetically or remotely. This is because once the dialysis bag was placed in the salt solution, the crystallization conditions could not be changed.

Example 2 Crystallization of Ribonuclease in Outer Space Ribonuclease S protein was purchased from Sigma Chemical Company. This protein is made up of 25% ammonium sulfate, 25% sodium chloride, 0%
. Dissolved in a 1 M solution of Na-acetate (24 mg / ml in pH 5.0) and dialyzed against the same solution overnight. At this concentration, protein crystallization is about 75% total salt saturation of the precipitant solution.
uration). At this level, the Con
trolled Crystallization System (DCCS)
Via dialysis with acetic acid and vapor diffusion in hanging drops
on in hanging drops). All solutions are
Standard 25% saturated ammonium sulfate and 0.1 M Na-acetate, pH 5.
0 was included. In all cases, the only changed precipitant was sodium chloride. Crystallization generally occurs when the concentration of NaCl reaches 47-52% saturation. Preliminary hanging drop trials were performed on Limbro plates (from Hampton Research).
linbro plate). The reservoir was filled with a solution containing a precipitant at a concentration expected to give a precipitate. They were combined with 250 μl of saturated ammonium sulfate, 40 μl of 2.5 M Na-acetate, 470-600 μl of saturated NaC
1 and water (up to a volume of 1 ml). NaCl
The amount of was selected by scanning over a wide range and finding the value that gave the best crystals. Hanging drops were formed by mixing equal volumes (5 μl) of protein solution and reservoir solution on siliconized cover slides (also from Hampton Research). The drops were kept at room temperature (24 ° C.). The process of vapor diffusion in a closed system gradually increased the salt concentration in the drop to the level of the reservoir and nucleation occurred. Other trials were performed using the DCCS of the present invention. In the method used in this example, the source syringe was filled with a level of precipitant solution at which crystallization was expected. The reservoir was filled with a lower level of a similar solution. It may contain standard components and only 35-40% sodium chloride. This concentration is too low to allow nucleation of ribonuclease S crystals. Also, these units were maintained at room temperature throughout the trial. As the motor pushed the source syringe plunger, the contents of the syringe were transferred to a reagent reservoir in the crystallization chamber, and the precipitant concentration in that reservoir was gradually increased to the concentration of the source syringe solution. The precipitant in the reagent solution in the crystallization chamber was then dispersed across the dialysis membrane into the reaction chamber, where the precipitant concentration reached the precipitant concentration of the reagent solution, and nucleation occurred. .

As expected, both methods produced similar crystals and were reproduced. At a sodium chloride concentration of about 53% saturation, small crystals may appear slightly overnight. These resulted in a size of about 0.1 mm and usually did not have a standard crystal shape. Their ends were not straight. Probably because it grew too fast. If nucleation is too rapid, the result is that it has a sub-standard morphology,
Too many undersized crystals. A better precipitant concentration is about 47-
50% sodium chloride. At these levels, about 1
Obtain ~ 5 crystals, some of which grow beyond 1 mm, which is ideal. Several more crystals were obtained with the DCCS unit. because,
This is because the volume of the protein chamber is much larger than the volume of the hanging drops. The 50 μl chamber in this unit contains 1.2 mg of protein whereas 10
The μl hanging drop contained only 0.12 mg of RNase S protein.

(Setup of Space Experiment) Final D concerning the STS-95 mission of Space Shuttle Discovery
For CCS preparation, filling was similar to filling in the laboratory trial. Four D
The CCS unit is connected to a Spa module using a bay module.
sce Hab, and 6 DCCSs, the ground Spa
sce Hab facility (Cape Canaveral, Florida). Of the six above-ground units, four were positive controls and were placed in a 24 ° C. incubator. The other two units were taken as spare units and placed outside the incubator on a laboratory bench. These were kept at about 24 ° C. The conditions in the source syringe for the flight unit were 47% sodium chloride in the F00 unit, F
01 was 50%, F02 was 53%, and F03 was 53%.
The starting reservoir was then filled with 35% sodium chloride. Six ground units were charged as follows: in the incubator, 47% sodium chloride at G00, 47% at G04, 50% at G01, and 53% at G02, and 50% at G03, and at the incubator. Outer 2
53% in one. The reservoir was initially filled at 35%. The length of the STS-95 mission was 10 days. The computer program was activated and the syringe was run for the first 3.5 days of the mission. Thus, the solution containing the protein was allowed to reach the expected nucleation point over 3.5 days and continued to grow the crystal over the remaining time.

Crystal Recovery, Photographs, Analysis and Mounting After the mission, the DCCS unit was then shipped by sea from Florida to the Center for Ad, Gaithersburg, MD.
advanced Research in Biotechnology (CAR
Transported to B) where it was opened. These crystals were counted through the membrane using a microscope. First, these crystals were photographed using a Nikon FX camera through a dialysis membrane while remaining in their growth environment. This allows for a comparison of the location in the chamber where the crystal was grown, as well as the appearance of the crystal before it was moved if damage occurred during the process. Next, this step:
Removing the crystals from the original protein chamber and placing the crystals in the artificial mother liquor in hanging drops. This mother liquor solution contains 60% sodium chloride,
Consists of 24% ammonium sulfate, and 1M sodium acetate. The crystals floating in the growth chamber were simply inverted onto the cover slide and poured out of the chamber. However, the crystals that grew stuck to the various walls and membranes of the chamber gently detached from the surface with the fiberloop and had to be physically put into hanging drops. At this point, the size of the crystals was measured using a microscope. In addition, observation of morphology and defects,
Recorded. Photos were then again taken using the same camera during this hanging drop environment and transferred to a digital image file. Once well recorded on the film and stabilized in solution, these crystals were used for X-ray diffraction analysis, 1.0 mm
Mounted on a siliconized glass capillary.

(Results) Upon unloading of STS-95, ribonuclease crystals were found in all DCCS units (except for two spare units that did not produce any crystals). As observed, the crystals maintained in the protein chamber showed little difference in the two growth environments. As expected, approximately the same number of crystals were present in each of the corresponding ground and space units. The amount of crystals obtained in both environments followed the expected curve (see Table 1). The higher the precipitant concentration, the more nucleation and the smaller the crystals. Crystals grown in space were approximately equal in size to their terrestrial growth counterparts. The crystal yield (a function of the protein actually incorporated into the crystal) was about the same. These two major differences were discovered when the recovery time came. A common problem usually occurred in ground units; crystals grown on the ground adhered to the walls and chamber membranes and were difficult to remove without damage. For all four ground units, about 80% of the crystals settled, and in particular, the largest and best crystals were the most difficult to recover. Crystals grown under microgravity are about 80% loose in their growth solution and
It was floating. The crystals were "poured out" onto the cover glass with little or no need for touch.

[0062]

[Table 2] Crystals obtained from ground and in space are generally similar in appearance, but there are some differences. Crystals from both environments typically had six well-developed faces with two slightly rounded tips. Because crystals on the ground adhere more often to the walls, they tend to have less integrity, ie, less than perfect shape. Conversely, crystals in space did not stick and floated freely, so they had a better and more uniform overall shape. However, due to its suspension, this space crystal, in some cases, had minor edge and tip damage, but did not significantly interfere with their analysis.

The computer program was running and completed as expected. All source syringes were empty and the drain syringes were full.

(Discussion) This mission achieved all of its original goals and was successful. Large crystals were obtained from both space and ground experiments. Topographic analysis required at least 0.5 mm of crystals, and some crystals from both environments and various precipitant conditions were recovered at this size. The size and number of RNase S crystals followed the size and number observed in preliminary experiments (crystal size and number increased with increasing salt concentration and decreasing in size) (see Table 1). See).

The main observed difference was that while the crystals adhered to the walls of the protein chamber in the ground unit, most of the crystals grown under microgravity were loose crystals. If crystals cannot be recovered from these growth environments, size and quality are not a problem. This ground-grown crystal grows much like a crystal grown in space and captures it on film. However, the damage that occurred during recovery further reduced the crystals in quality for analytical purposes. Cracks often occur and broken corners have been a common problem when breaking crystals away from the growing surface.

All publications, patents and applications mentioned in the above detailed description are herein incorporated by reference. Nothing herein is to be construed as an admission that the present invention does not provide such disclosure by prior art to the preceding examples. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific embodiments, it is to be understood that the invention as claimed should not be limited to such specific embodiments, and that those skilled in the art to which the invention pertains will be described. Various modifications of the described methods for carrying out the invention which are obvious are intended to be within the scope of the following claims.

[Brief description of the drawings]

FIG. 1 is a detailed schematic diagram of a crystallization apparatus of the present invention.

FIG. 2 is a schematic diagram of a controller for controlling a crystallization apparatus.

FIG. 3 is a detailed schematic diagram of an embodiment of a crystallization chamber, wherein a dialysis membrane separates the reaction solution of the upper assembly chamber from the lower reaction solution.

FIG. 4 is a detailed top view of an embodiment of a crystallization chamber, showing attached ionic strength, pH, and temperature probes.

FIG. 5 schematically shows a series of crystallization chambers, wherein the crystallization chambers are connected to each other by small diameter tubing, and the first and last crystallization chambers are connected via tubing. , A source reservoir and a drain reservoir (eg, a syringe).

FIG. 6 schematically illustrates a row of crystallization chambers connected to source and drain reservoirs via a manifold.

FIG. 7 schematically illustrates a 2-D matrix of a crystallization chamber, the crystallization chamber in this row being connected via a manifold to a pair of source and drain reservoirs. And the crystallization chambers of this column are connected via manifolds to different pairs of source and drain reservoirs.

FIG. 8 schematically illustrates a series of crystallization chambers in the bore of a magnet, which can produce an effective gravitational field of 0 to 2 times the gravity at the surface.

[Procedure amendment]

[Submission date] April 27, 2001 (2001.4.27)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0044

[Correction method] Change

[Correction contents]

FIG. 5 schematically shows a series of crystallization chambers 91 connected together by small diameter tubing 92. The first and last crystallization chambers are connected via tubing to a source syringe 93 and a drain syringe 94 containing a reagent solution of a selected concentration. The source and drain syringes or "reservoirs" shown in FIG. 5 correspond to the syringes 16 and 18 of FIG. 1, respectively, and perform similar functions. In one embodiment, all crystallization chambers in series initially contain the same reactant and reagent solutions. Each trace of solution is injected from the source syringe into the first cell in series, an equal volume of solution is transferred from each cell to tubing leading "downstream" to the next adjacent series of cells, and the last cell To the drain syringe. The reagent solution containing a high concentration of precipitant injected from each trace source syringe was mixed with the reagent solution in the first cell and diluted, and thus was transferred from the first cell to the second cell. The reagent solution has a lower concentration of precipitant than the trace reagent solution injected into the first cell. Dilution of the transferred reagent solution occurs in each subsequent cell in series in a similar manner. Thus, the rate of increase of the precipitant concentration is fastest in the crystallization chamber adjacent to the source reservoir and decreases continuously in each successive chamber in series. For complete control of the precipitant concentration in the cells, it is important that the reagent solution does not flow or diffuse between the cells except during controlled movement. Very small diameter tubing is used to connect the cells to prevent “cross flow”. Alternatively, a valve that is open only while liquid is being transferred between cells may be inserted into the line of tubing 92 between cells.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0045

[Correction method] Change

[Correction contents]

FIG. 6 schematically shows an embodiment of the present invention. The reagent solution in an embodiment of the present invention is transferred from the source reservoir 93 via the manifold 95 to the plurality of crystallization cells 91, each of the plurality of crystallization cells may be changed in an initial reactant and / or reagent solution. . This reagent solution is transferred from the crystallization cell 91 to the drain storage tank 94 via the manifold 96. For this embodiment, the crystallization process can be monitored in a set of crystallization chambers with different initial conditions, as the concentration of one or more precipitants changes simultaneously in all of the cells in a regulated manner.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0046

[Correction method] Change

[Correction contents]

The present invention also includes configuring a crystallization chamber in a two-dimensional or three-dimensional matrix according to the techniques described above to analyze crystallization under a number of different dynamically controlled conditions. FIG. 7 schematically shows a 2-D matrix of the crystallization chamber. Two different solutions (each solution containing one or more precipitants) are pumped in different directions through the rows and columns of the cell to create independently controlled concentration gradients of the precipitants. The paired source and drain syringes operate in tandem, with different source / drain pairs (101/102 and 103/104) operated continuously. Cross-flow between cells is prevented by connecting the cells using small-bore tubing 92 and, if necessary, connecting valves in the line between cells that are open only when fluid is transferred. Is prevented.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0047

[Correction method] Change

[Correction contents]

In a similar manner, the effect of crystallization by changing the concentration of one or more additional precipitants in multiple crystallization chambers is to reduce the crystallization chambers of two or more matrices (eg, “stacks”). Recognized by joining, one or more gradients of precipitant concentration may be established to form a third dimension. A pair of source and drain syringes controlling the flow of additional reagent solution into and out of the crystallization chamber of the first matrix and from the chamber of the last matrix were operated in tandem and described above with respect to the syringe pair of the 2-D matrix. In this manner, the other pairs of syringes are sequentially activated. Of course, there are countless modifications and combinations of the above-described configurations of the invention that can be used to analyze crystallization. Analysis of crystallization is, for example, by using multiple and independently controlled source syringes connected to the same or different cells in a given dimension, and by changing the initial conditions in the crystallization chamber. Such modifications of the methods and apparatus described herein are readily within the ability of those skilled in the art to produce molecular crystals and are included in the present invention.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0051

[Correction method] Change

[Correction contents]

In order to grow crystals under different effective gravity ranges, the vertical assembly of the crystallization chamber 71 supported by the support strip 72 may be provided with a high magnetic field (eg, 16T) hole 73 as shown in FIG. Is placed within. Each crystallization chamber 71 consists of a reactant reservoir with a volume of about 1 ml. Reactant reservoir in communication with the reagent chamber through the dialysis membrane, as described above (volume of a few mm ^3). Thus, each crystallization chamber has a different g _eff value and a different magnetic field value. For a given magnetic field value, the crystallization chamber can be placed in the g _eff = 0 condition by adjusting the entire stack. It is important to note that the stack does not levitate. Rather, for g _eff = 0 with little force over a distance less than 1 mm, there are microcrystals of the reactant in a particular cell. Each chamber in the stack _attempts a step change in g _eff for the position as shown in FIG. The chamber above the center of the magnet has g _eff <g and below the center of the magnet g _eff > g. The center chamber shows a control experiment when a magnetic field is present (where g _eff = g). The first crystallization chamber 71 shown in FIG. 8 is connected to a reagent solution source reservoir by an inlet tube 74. Small diameter tubing connects each crystallization chamber to the next in the series and the last chamber in the series is connected by a return tube 75 to the drain reservoir. The configuration of this crystallization chamber is the same as that shown in FIG. By controlling the flow of the reagent solution containing one or more precipitants through the series chamber, a concentration gradient of the precipitant can be established across the crystallization chambers in series. This measure allows for crystal growth in a multifactorial environment where both the precipitant concentration and the effective gravitational field change. Further, the total magnetic field can also vary. The total magnetic field can vary between about 16 and 33 Tesla. This shifts the points g _eff = (2 to 6) g and g _eff = 0 further away from the center of the magnet. For fields smaller than 16T, the g _eff = 0 condition cannot be maintained, but smaller changes can be obtained for the condition 2g <g _eff <g.

[Procedure amendment 6]

[Document name to be amended] Drawing

[Correction target item name] Fig. 5

[Correction method] Change

[Correction contents]

FIG. 5

[Procedure amendment 7]

[Document name to be amended] Drawing

[Correction target item name] Fig. 6

[Correction method] Change

[Correction contents]

FIG. 6

[Procedure amendment 8]

[Document name to be amended] Drawing

[Correction target item name] Fig. 7

[Correction method] Change

[Correction contents]

FIG. 7

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF terms (reference) 4G075 AA22 BA10 BB10 BD15 CA51 CA65 CA72 DA13 EA01 EB01 EC06 ED13 4G077 AA01 BF05 CB02 CB10 EG30 EJ06 HA20 [Continuation of summary]

Claims (1)

[Claims] 1. An apparatus for crystallizing a molecule, comprising: a reactant solution containing the molecule to be crystallized and at least one crystallization arranged to contain a reagent solution containing at least one precipitant. A chamber wherein the solutions contact and are separated by one of a dialysis membrane containing pores and a permanent sieve, wherein the pores are separated from one solution to another by the at least one solution.
Large enough to allow the passage of two precipitants, but not large enough to allow the passage of the molecules to be crystallized; at least one first reagent solution reservoir and at least one second reagent solution reservoir A first and second reagent solution reservoir, wherein the first and second reagent solution reservoirs are arranged to receive respective reagent solutions containing the at least one precipitant; At least one first communication means for communicating the reagent solution in the crystallization chamber; at least one second communication means for communicating the second reagent solution reservoir with the reagent solution in the crystallization chamber. Communication means; at least one first transfer means for selectively transferring a solution between the first reagent solution storage tank and the reagent solution in the crystallization chamber; the second reagent solution storage tank and the crystal Chamber At least one second transfer means for selectively transferring a solution to and from a reagent solution; and the first transfer means and the second transfer means in cooperation with the first transfer means and the second transfer means. Interlocking means for reversibly transferring an equal volume of reagent solution into and out of said crystallization chamber by means of two transfer means.