Abstract:
A crystallization tray includes a plurality of crystallization cells, each cell having a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use as a temporary cryogenic holding area for a crystallized substance and/or a sample holding area, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance. A related method for forming macromolecular crystals includes dispensing an equilibrating solution in the reservoirs, dispensing a plurality of macromolecular solution droplets in the sample drop receptacles, covering the cells with a cover; and crystallizing the crystallizable substance by vapor diffusion.

Description:
FIELD OF THE INVENTION 
     The present invention relates, in general, to crystallization trays, and, in particular, to trays for forming diffraction-quality macromolecule crystals by vapor-diffusion techniques. 
     BACKGROUND OF THE INVENTION 
     Supersaturated solutions of biological macromolecules (e.g., proteins, nucleic acids) under defined conditions form macromolecular crystals. Macromolecular crystals have been used in the biotechnology/pharmaceutical industry for many purposes. For example, three-dimensional structural models of macromolecule structures derived from X-ray crystallography are used to design new drugs and compositions in pharmaceutical and agricultural research; crystallization steps are utilized in purification/manufacturing processes of biotechnology-derived products; and crystalline complexes are used for controlled-release drug formulations and agricultural products, such as, for example, herbicides and insecticides. 
     One of the first and most important steps in the X-ray crystal structure determination of a target macromolecule is to grow suitably large, well-diffracting crystals of the macromolecule. Compared to the technological advances achieved in making, collecting, and analyzing X-ray diffraction data more rapid and automated, crystal growth has become a rate-limiting step in the structure determination process. 
     Vapor diffusion is the most widely used technique for crystallization in modern macromolecular X-ray crystallography. In this technique, a small volume of the macromolecule sample is mixed with an approximately equal volume of a crystallization solution to form a sample solution. A drop of the sample solution is sealed in a cell with a much larger reservoir volume of the crystallization solution. The drop is kept separate from the reservoir of crystallization solution either by hanging the drop from a cover slip (“hanging drop” technique) or by sitting the drop on a pedestal (“sitting drop” technique) above the level of the crystallization solution in the reservoir. Over time, the crystallization drop and the equilibrating solution equilibrate via vapor diffusion of volatile chemical species. Supersaturating concentrations of the macromolecule are achieved, resulting in crystallization of the macromolecule sample in the drop. 
     Typically, several hundreds of experiments must be performed before conditions are found to produce high-quality crystals. Some of the conditions that are screened to determine the optimal conditions for crystal growth are pH, temperature, concentration of salts in the crystallization drop, concentration of the macromolecule to be crystallized, and concentration of the precipitating agent (of which there may be hundreds). Testing numerous combinations of conditions that affect crystal growth, by means of hundreds to thousands of crystallization experiments, eventually leads to the optimal conditions for crystal growth. Consequently, the ability to rapidly and easily generate many crystallization trials is important in determining the ideal conditions for crystallization. 
     Crystallization trays have been developed in the past in an effort to permit the efficient testing of numerous combinations of conditions that affect crystal growth. A problem with past trays is that the number of crystallization cells or chambers in a tray were too large and too few (e.g., 24 cells), causing the trays to be too large and/or limiting the number of crystallization experiments performed on a given tray. Another problem with past trays is that the crystallization cells did not have good viewing characteristics for viewing the crystallization process under a microscope or by using an imaging system. The cells often included solution reservoirs or sample receptacles that were too curved and/or were not clear, inhibiting viewing the crystallization process. A further problem with many past trays is that they are not sized to the standards set forth by the Society for Biomolecular Screening (SBS). Consequently, these trays can not be easily used with automated robotic equipment designed for trays meeting these standards. A still further problem with some past trays is that the trays are not formed as an integrated, single piece. For example, one tray in the past included multiple rows of crystallization cells that were removable from the tray base during use. The manufacture of multiple pieces made this tray expensive and susceptible to the cell rows accidentally dislodging from the tray base. Another problem with many trays in the past is that they are not appropriately designed for more general crystallography. Trays used for the hanging drop method have the disadvantage of not being easily used in automated processes because they require an automated mechanism for inverting the surface on which the drop is placed, such as a cover slip. But, many trays used for sitting drops are also not appropriate for maintaining the appropriately shaped drop. For example, in some crystallography trays, the floor of the sample solution receptacle is too flat and/or the floor includes sharp, angled corners. This may cause the sample solution to smear, cause a thin film to form, and/or cause the drop to migrate to the corner(s), inhibiting or preventing successful crystallization. If the drop migrates to a corner or corners, consistent visualization with a microscope is difficult because a consistent place may not exist in the cell for predicting where the sample is for visualization. In addition, commercially available trays do not provide for a simple way to seal individual cells without requiring the use of individual cover slips. 
     SUMMARY OF THE INVENTION 
     An aspect of the invention involves a crystallization cell including a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use as a temporary cryogenic holding area for a crystallized substance, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance. 
     Another aspect of the invention involves a crystallization tray. The crystallization tray includes a plurality of crystallization cells, each cell having a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use, for example, as a flat sample holding surface, and/or as a temporary cryogenic holding area for a crystallized substance, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance. 
     An additional aspect of the invention involves a crystallization tray including a base with a plurality of rectangular crystallization cells, a top wall, and a rectangular ridge extending upwardly from the top wall and delineating each cell. The ridge is adapted to support a cover for isolating each cell from ambient and from each other. The cover is preferably clear; preferred covers are self-adhesive, such as adhesive tape or plate sealer (available from, for example, 3M). The cover may also be one or a plurality of cover slips, covering either all of the crystallization cells, individual cells, or groups of cells such as rows or columns. Where the cover is not adhesive, the ridges of the tray could be greased with, for example, petroleum jelly, sealing medium, or grease, or gaskets could be used to attach the cover. 
     A still further aspect of the invention involves a method for forming macromolecular crystals. The method includes providing a macromolecule crystallization tray having a plurality of crystallization cells, each cell including a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use as a flat sample holding surface and/or as a temporary cryogenic holding area for a crystallized substance, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance; dispensing an equilibrating solution in the reservoirs, dispensing a plurality of macromolecular solution droplets in the sample drop receptacles, covering the cells with a cover; and crystallizing the crystallizable substance by vapor diffusion. 
     These and further objects and advantages will be apparent to those skilled in the art in connection with the drawing and the detailed description of the preferred embodiment set forth below. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective view of an embodiment of a macromolecule crystallization tray. 
     FIG. 2 is a top plan view of the tray illustrated in FIG.  1 . 
     FIG. 3 is a partial, cross-sectional view of the tray taken along line  3 — 3  of FIG.  2 . 
     FIG. 4 is an enlarged cross-sectional view of the area denoted as  4  in FIG.  3 . 
     FIG. 5 is a perspective view of a sliced section of a well of the tray illustrated in FIG.  1 . 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     With reference to FIG. 1, a crystallization tray  100  constructed in accordance with an embodiment of the invention will now be described. The tray  100  is especially useful in crystallizing biological macromolecules such as proteins and nucleic acids; however, the tray  100  may be used to crystallize any crystallizable substance. Some general material properties of the tray  100  will first be described, followed by a description of the elements of the tray  100  and the tray  100  in use. 
     The tray  100  preferably has a single-piece, integrated construction and is manufactured using an injection molding process. The tray  100  is preferably made from an optically clear, plastic material such as, but not by way of limitation, a clear polystyrene material or polypropylene polymer. An optically clear, inert, plastic material allows crystal growth to be viewed under a microscope. Crystal growth may also be viewed using an imaging system, such as, for example, a video camera operably linked to a computer having a computer monitor that can display the image. The plastic material of the tray  100  preferably is a low-wettability material having a relatively high contact angle with respect to water so that the solution of the sample to be crystallized will tend to form discrete drops when placed in contact with the crystallization tray  100 . The plastic material selected should be moldable so that the inner surfaces of the tray  100  that come into contact with the macromolecule solution have a smooth texture. The material used should be resistant to chemicals such as methyl pentane diol, organic acids and alcohols, and should be stable for long term storage in pH 3-10 solutions. 
     The tray  100  includes a rectangular base  110  with an array of crystallographic cells  120  and a rectangular skirt  130  joined with the base  110  through a step  140 . The rectangular dimensions of the base  110  are slightly smaller than the rectangular dimensions of the skirt  130 , and the undersurface of the rectangular skirt  130  is open and hollow, allowing for stacking of multiple trays  100 . 
     The base  110  includes a first side wall  150 , a second side wall  160 , a first end wall  170 , a second end wall  180 , and a top wall  190 . Where the first side wall  150  and first end wall  170  would normally intersect, the base  110  includes a single beveled or cut corner  200  for orienting the tray  100  (e.g., always knowing which way is up) and to denote a first cell  210  in the array. In a preferred embodiment, the tray  100  includes ninety-six cells  120  organized in twelve columns denoted respectively by the numbers 1-12 and eight rows denoted respectively by the letters A-H. The beveled corner  200  preferably indicates the position of the A-1 cell (i.e., the first cell, the cell in the first row and first column, or the cell located in column A, row 1). The top wall  190  preferably includes three knobs  220  that are used for orienting the tray  100  in the imaging system. 
     With reference to FIGS. 2-5 and especially FIGS. 3-5, the crystallization cells  120  will now be described in more detail. Each cell  120  generally includes a reservoir  230 , a shelf  240 , and a sample receptacle  250 . The cell  120  is generally defined by opposite side walls  260 , a first end wall  270 , a second end wall  280  comprised of a lower wall portion  290  and an upper wall portion  300  joined by the shelf  240 , and a bottom wall  310 . 
     The side walls  260  and end walls  270 ,  280  terminate at the top of the cells  120  in ridges  320 . The ridges  320  include top walls  330  that define a second plane that is parallel with and extends above a first plane defined by the top wall  190  of the base. When a cover is applied to the top of the tray  100 , the cover is supported by the ridges  320 . The cover seals each cell  120  along the cell&#39;s surrounding ridge  320  and isolates the cell  120  from adjacent cells  120 . The space between adjacent ridges  320  is preferably just wider than a blade of a cutting instrument for selectively cutting the cover around one or more select cells  120  in the tray  100  to access the crystallized substance in the cell(s)  120  without disturbing the cover for adjacent cells  120 . Widening the ridges  320 , causing the space between adjacent ridges  320  to be narrower, creates a better top wall sealing surface around the cells  120  for the cover and creates a better, more defined cutting path for the blade of the cutting instrument. 
     The reservoir  230  is preferably a generally rectangular block-shaped void. In a preferred embodiment, the reservoir  230  is sized to accommodate approximately 100 uL of equilibrating solution. In alternative embodiments, the reservoir  230  may have a different configuration and/or may be sized to accommodate other volumes of equilibrating solution. 
     With reference to FIG. 4, the sample receptacle  250  is preferably cup-shaped with a flat bottom surface  340 . The flat bottom surface  340  allows for optical clarity for viewing the drop, and the size of the sample receptacle  250  helps to keep the sample solution held together in a tight drop and prevents spreading of the drop. This makes the sample receptacle  250  appropriate for both macromolecule crystallography and for more general crystallography. This also keeps the drop centered, in a consistent position for visualization with a microscope. The sample receptacle  250  is preferably sized to accommodate a 2 μl sample drop. Sizing the sample receptacle this small is advantageous because it limits where the sample is in the receptacle, making it easier to locate for visualization, removal, etc. The flat bottom surface  340  of the sample receptacle  250  is curved at the edge where it meets the side wall of the sample receptacle  250 , allowing for a crystallization sample retrieving device (e.g. nylon fiber microloop connected to shaft) to be used to smoothly scoop the crystal out of the well without jarring the crystal against the side wall. 
     Preferably, the ratio of the reservoir volume to the sample receptacle volume is at least large enough to create an appropriate gradient to drive the concentration of the protein drop high enough to cause the protein to crystallize. Those of ordinary skill in the art are aware that the larger the reservoir volume, as compared to the sample receptacle volume, the steeper the gradient will be. This allows for more reproducibility, and the easiest way to determine the likeliness that the crystallization step has been driven to completion. The ratio, however, is preferably within a practical range that allows for high-throughput crystallization. Thus, the ratio of the reservoir volume to sample size may be, for example, at least about 500:1, preferably at least about 100:1, preferably at least 75:1, more preferably at least about 50:1, preferably at least about 40:1, and preferably at least about 25:1. In one preferred example, the reservoir is sized to accommodate an equilibrating solution volume of about 100 μl and the sample receptacle is sized to accommodate a sample volume of about 2 μl. 
     With reference back specifically to FIG. 2, the shelf  240  carries the sample receptacle  250  adjacent one of the side walls  260  and includes a flat upper surface  350 . A majority portion  360  of the flat upper surface  350  is located between the sample receptacle  250  and opposite side wall  260 . As used herein, “majority” means greater than 50%. When the tray  100  is oriented in the standard position shown in FIG. 2 so that the A-1 cell  210  is the upper-left corner of the tray  100  (i.e., beveled corner  200  is upper-left corner of tray  100 ), the majority portion  360  of the flat upper surface  350  is located away from the user with respect to the sample receptacle  250  It has been determined by the inventor of the present invention that locating the flat surface of the shelf  240  in this position is ideal for right-handed users (right-handed users are statistically more common than left-handed users) because it allows the crystallized sample to be easily moved forward onto the flat surface of the shelf  240  with a crystallization sample retrieving device (e.g. nylon fiber microloop connected to shaft). It has been determined that this is more convenient for the user than drawing or dragging the crystallized sample rearward, towards the user. Further, when the tray  100  is oriented in the position shown, the shelf  240  is located on the left side of the cell  120 . Because the crystallization sample retrieving device is typically operated by the right hand of a right-handed user, locating the shelf  240  on the left side of the cell  120  allows for easier access to the sample receptacle  250  and shelf  240  with the crystallization sample retrieving device. The majority portion  360  of the flat upper surface  350  of the shelf  240  may serve as a cryoprotection holding area for the crystallized sample between crystallization and X-ray diffraction. It is important to cryoprotect the crystallized sample after crystallization and before X-ray diffraction. Instead of having to remove the crystallized sample from the cell and cryoprotect outside of the cell, this can be done in the holding area, within the cell  120 . Providing a cryoprotection holding area is important because once the crystallized sample is removed from the cell  120 , it may quickly deteriorate in the air because a macromolecule crystal is about 50% solvent and prone to dehydration. 
     The majority portion  360  of the flat upper surface  350  may also hold a sample drop in addition to, or instead of, the receptacle  250 . This may be desirable for performing, in each cell, two different experiments with the same type of sample or different types of samples, or a single experiment with the sample only placed on the shelf  240 . Because the shelf  240  is flatter than the receptacle  250 , the drop spreads out more when placed on the shelf  240 , increasing the surface-to-volume ratio of the drop, changing the kinetics of the equilibrium experiment. 
     In use, the tray  100  is oriented in the standard position shown in FIG. 2 so that the A-1 cell  210  is the upper-left corner of the tray  100  (i.e., angled corner  200  is upper-left corner of tray  100 ). Each reservoir  230  is carefully filled with a selected equilibrating solution. Different equilibrating solutions can be added to each of the reservoirs  230  if this is desired. Typically, aqueous mixtures of buffer, salts, and precipitants such as polyethylene glycol or ammonium sulfate are used as precipitating agents in the equilibrating solution. This solution may contain other components such as organic molecules or other additives. Preferably, approximately 100 μl of equilibrating solution is added to each of the reservoirs  230 . In alternative embodiments, the amount of equilibrating solution added to each of the reservoirs  230  may be greater than 100 μl, less than 100 μl, different amounts of equilibrating solution may be added to the reservoirs  230 , and/or different types of equilibrating solution may be added to the reservoirs  230 . 
     Following the addition of the equilibrating solution to the reservoirs  230 , a selected macromolecule (e.g., protein) solution drop is deposited within each sample receptacle  250 . The drop is preferably approximately 2 μL and includes a protein in a buffered salt solution containing a lower concentration of the same precipitating agent used in the equilibrating solution. Usually, a concentrated protein solution is mixed with the equilibrating solution to obtain the final total volume of, for example, 2 μl. The ratio of protein to equilibrating solution may be varied, and may be, for example, 1:1. 
     It is to be understood that the equilibrating solution as well as the macromolecule solution drops may be added to the cells  120  either by hand or by a sophisticated automated pipetting apparatus which is readily commercially available. Because of the novel construction of the tray  100  and the systematic placement of the reservoirs and receptacles, the tray  100  is readily adaptable to most commercially available pipetting systems. 
     Once the equilibrating solution and protein drops have been added to the apparatus in the manner described, the cover is carefully placed over the tray  100  so that the top walls  330  of the ridges  320  are positively sealed relative to atmosphere. The tray is designed to provide effective sealing between the top walls  330  and the under surface of the cover, using adhesive tape or a plate sealer. Alternatively, a layer of grease, such as silicon grease, or petroleum jelly, may be applied manually or automatically to the top walls  330  of the ridges  320 , and some other clear, impermeable layer plated over the tray. 
     Instead of a sitting-drop technique, in an alternative embodiment, a hanging-drop technique may be used for applying the protein drops to the cells  120 . The protein drops may be applied to an undersurface of the cover (with the undersurface face up), and the cover may be inverted and the undersurface sealed against the top walls  330  of the ridges  320  so that the protein drops are hanging into the cells  120 . 
     With either technique, each protein drop is positively sealed within each reservoir cell  120  and can equilibrate against the particular equilibrating solution that was earlier deposited into the particular reservoir  230 . Since the starting concentration of precipitating agent is always higher in the reservoir  230  than in the protein drop, once the cell  120  is sealed, water will diffuse from the protein drop to the reservoir  230  until the concentration of precipitating agent at equilibrium is the same in the drop as in the reservoir  230 . In general, this diffusion results in a controlled steady increase in the concentration of both the protein and precipitating agent within the drop which forces the protein to come out of solution, hopefully as a crystal. Or, in the instance where there is a dilution effect, where the buffer that is in the protein sample is more concentrated than in the reservoir, the protein is said to “salt into solution” and the crystallization drop tends to grow. 
     Because the tray  100  is made of an optically clear material, the crystallization of each sample may be viewed using a microscope or other visualization apparatus. 
     Following crystallization of the protein drops, the cover of the tray  100  is removed. As described above, a blade may be used between the ridges  320  to cut the cover into one or more cover segments that may be individually removed from the top wall  330  of the ridge  320 . After removal of the cover segment(s), the crystals are preferably moved onto the majority portion  360  of the flat surface  350  of the shelf  240  with the crystallization sample retrieving device. The flat surface  360  may serve as a cryoprotection holding area for the crystal. A cryoprotectant may be added to the shelf  240  before or after, preferably before, the crystallized sample is moved to the majority portion  360  of the shelf  240 . The crystallized sample may then be swept through the cryoprotectant. As indicated above, moving the crystallized sample to the cryoprotection holding area before removing the sample from the cell  120  helps preserve the crystallized protein. From the flat surface  360 , each crystal may be removed from the cell  120  with the crystallization sample retrieving device and analyzed through X-ray diffraction. In alternative embodiments, the crystals may be analyzed within the tray  100 , the crystals may be removed directly from the receptacles  250  or removed in another manner, and/or the crystals may be analyzed by a technique other than X-ray diffraction. 
     Because the crystallization tray  100  includes a rectangular array of ninety-six crystallization cells  120 , the user may simultaneously screen up to ninety-six different combinations of factors that affect crystallization. Additionally, the number of factors that can be simultaneously tested can be increased by placing an additional sample drop on the flat majority portion  360  of the shelf  240 . 
     Thus, the crystallization tray  100  offers many advantages over crystallization trays in the past, some of which are summarized below. The shelf  240  provides a cryoprotection holding area and additional flat sample holding surface for performing an additional and/or different type of crystallization experiment in the cell  120 . The shape of the sample receptacle  250  maintains the sample drop held together, in a tight drop, and prevents spreading of the drop. The small size of the sample receptacle  250  makes it easy to locate the crystallized sample in the receptacle  250 . The configuration of the shelf  240  makes it ideal for right-handed users to access, move, and/or remove the crystallized sample. The three knobs  220  on the top wall  190  of the tray  100  help for orientation and calibration of the tray  100 . The cut corner  200  also helps for orienting the tray  100  and quickly identifying the first cell in the array. The raised, rectangular ridges  320  provide a sealing support surface for the cover(s), allow the cover(s) around individual cells  120  to be selectively removed without disturbing the cover(s) on adjacent cells  120 , and allow access between ridges for cutting away the cover(s) around one or more selected cells. The flat bottom  340  of the sample receptacle  250  allows for ease of imaging. The curvature of the angle between the walls and the bottom of the sample receptacle  250  is suitable for sweeping out the crystallized sample with a mounting loop. 
     It will be readily apparent to those skilled in the art that still further changes and modifications in the actual concepts described herein can readily be made without departing from the spirit and scope of the invention as defined by the following claims.