Patent Application
20110060126
The present invention relates to improvements in the field of crystallography. In particular, this invention relates to a new method or strategy for optimizing crystallization conditions of a given substrate. The invention also relates to a new multi-well plate for carrying out the optimization of the crystallization conditions of the substrate.
During the last decade, the technical aspect of structural biology has been greatly simplified by high-throughput methods, applied from protein expression up to data collection. Even with that gained advantage, Crystal Growth remains an important challenging step of crystallography. As automation processes are becoming routine in laboratories, increasing the number of performed crystallization experiments on a day to day basis, there is still a constant decrease in the success value of these experiments (# Structure solved/Experimental Setup).
In order to obtain crystals for protein 3D structure determination, crystallographers use well known strategies where a protein is initially screened against a wide array of conditions in order to determine a “hit solution” for a given protein. From this set of initial conditions, crystalline forms or “hits” are observed and several optimization rounds, centered on the initial condition producing the hit, are often necessary to get essential quality crystal.
The most popular and used optimization strategy is performed by varying components of the experimental chemical conditions and preparing grids around the initial hit (for a clear review, see McPherson, A. Crystallization of biological macromolecules. 1999. New-York: Cold Spring Harbor Library Press, 291-296). This approach allows to determine which factor influences crystallization of a particular protein and to what extent it can improve crystal quality. Many parameters can be varied in trying to optimize an initial hit. Such parameters are, for example, precipitant concentration, pH, type of buffer, salt ions, additives such as reducing agents, metal ions, inhibitors etc., protein (concentration, source, mutant etc.), and experimental conditions (temperature, methods, etc.).
However, when working with a new protein, even a very experienced crystallographer may have some difficulties selecting which factors are important and which are not.
The method using the “expanded grid” is a very well designed strategy of optimization but it constitutes a tedious and time consuming procedure. Also, rounds of optimization centered on an initial crystallization hit does not always bring the ultimate goal of getting a crystal since the hit may itself be the optimized condition corresponding to this particular chemical environment.
Optimization of crystallization condition is usually carried out by slight variations of the chemical environment around an initial hit. Such a process is tedious and time consuming since many questions must be asked in order to determine which factors must be varied first, how to apply the selected changes to initial hit and in what format. The crystallographer also has to determine if the variations brought to the parameters significantly vary initial crystallization hit and create a new condition and if the crystallization space around the hit is well covered. Therefore, it appears that the methods and strategies proposed so far do not provide efficient and rapid solution for the optimization of the crystallization conditions of a protein, and that new methods would be required.
Macromolecular crystallization keeps getting faster and easier to setup, but crystal growth still remains a trial & error process. It is rare that an initial screening alone provides high-resolution crystals. Many rounds of optimization are necessary to get diffraction quality crystals.
With automation and high-throughput techniques present in more and more laboratories, “mild results” in initial screenings of protein alone still points toward the fact that the methodology aspect of crystal growth needs a second look. In particular, the relationship between initial screening and optimization requires more attention.
It is therefore an object of the present invention to provide a method and an apparatus for optimizing crystallization conditions, which would overcome the above-mentioned drawbacks.
It is another object of the present invention to provide a method and an apparatus for rapidly and simply optimizing crystallization conditions of a substrate.
It is another object of the present invention to provide a method and for optimizing crystallization conditions of a substrate, which could be carried out in a single round after the determination of the hit solution.
According to one aspect of the invention, there is provided a multi-well plate comprising a plurality of wells, each well having therein a different crystallization media, each crystallization media varying according to at least two different parameters, a first parameter having at least one condition, and a second parameter having at least two different conditions, whereby said multi-well plate allows to facilitate optimization of crystallization conditions of a substrate.
The parameters may be for example selected from the group consisting of a buffer, pH of said crystallization media, salt, concentration of said salt, temperature of said crystallization media, additive, concentration of said additive, co-crystallization compound, concentration of said co-crystallization compound, alcohol, concentration of said alcohol, polymer, and concentration of said polymer.
In one embodiment of the invention, one of said parameters is the buffer. Each condition of said buffer parameter can be represented by a predetermined buffer that can be selected from the group consisting of Tris, Tris HCI, HEPES, Sodium HEPES, Imidazole, Sodium Citrate, Sodium Cacodylate and Sodium Acetate.
Alternatively, one of said parameters can be the pH of said crystallization media. Each condition of the pH can represents a different pH value to be tested.
One of said parameters can also be the salt. Each condition of the salt can thus represents a different salt, that can each comprise an inorganic or an organic anion, and an organic cation, or alternatively, an organic anion, and an inorganic or an organic cation.
The cation can be for example selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium and lithium, and the anion can be selected from the group consisting of formate, malonate, chloride, acetate, fluoride, bromide, nitrate and thiocyanate.
In one embodiment, one of said parameters is the concentration of the salt. Each condition of the salt concentration can thus be represented by a different concentration value of said salt.
In another embodiment of the invention, one of said parameters is the temperature of said crystallization media, where each condition of the temperature media can thus be represented by a different temperature to be tested.
In a further embodiment of the invention, one of said parameters is the additive, and thus each condition of the additive can be represented by a different additive, such as a reducing agent, a metal ion, an inhibitor or a detergent.
Still in one embodiment of the invention, one of said parameters is the concentration of said additive, where each condition of the additive concentration can thus be represented by a different concentration value of said additive to be tested.
In a further embodiment of the invention, one of said parameters is the ligand, where each condition of the ligand can thus be represented by a different ligand to be tested. For example, the predetermined ligand can be selected from the group consisting of ATP, ADT, NAD, NADP, NADPH, NADH.
In a further embodiment of the invention, one of the parameters is the concentration of the ligand, where each condition of the ligand concentration can thus be represented by a different concentration value of said ligand to be tested.
In a further embodiment of the invention, one of the parameters is the alcohol, where each condition of the alcohol can be represented by a predetermined alcohol to be tested. Examples of alcohol can be selected from the group consisting of methanol, ethanol, propanol isopropanol, methylpentanediol, hexanediol, and ethylene glycol.
In a further embodiment of the invention, one of the parameters is the concentration of said alcohol, where each condition of the alcohol concentration to be tested can thus be represented by a different concentration value of said alcohol.
In a further embodiment of the invention, one of the parameters is the polymer, where each condition of the polymer can thus be represented by a different polymer to be tested, such as PEG, polyethyleneimine and Jeffamine M-600.
In a further embodiment of the invention, one of the parameters is the concentration of said polymer, where each condition of the polymer concentration to be tested can thus be represented by a different concentration value of said polymer.
The crystallization media can thus vary according to at least two, preferably more than two and more preferably three different parameters, where a first parameter has at least one condition, and a second parameter has at least two different conditions, and a third parameter has at least one and preferably two, condition.
In another embodiment of the invention, the first parameter is the additive, said second parameter is the concentration of said additive, and said third parameter is the pH of said crystallization media. In still a further embodiment of the invention, the first parameter is the salt, said second parameter is the concentration of said salt, and said third parameter is the pH of said crystallization media.
The plate is a multi-well plate that can comprise any number of wells such as 3, 6, 24, 96, 192, 384, 768 or 1536 wells, and more preferably 96 wells.
In yet a further embodiment of the invention, there is provided a plate as defined above and comprising 96 wells, said first parameter being the salt and the conditions of said first parameter being 16 different salts, said second parameter being the salt concentration and the conditions of said second parameter being 2 different concentrations, and said third parameter being the pH and the conditions of said third parameter are 3 different pH values.
The crystallization media used in the plate can either be a solution or a gel. The plate preferably further comprises a cover disposed on said wells to seal them.
The plate can be of the hanging-drop crystallization type of plate, the plate further comprising a cover for sealing said wells, or of the sitting drop crystallization type of plate.
Each well of the plate may comprise a crystallization media reservoir adjacent to a substrate well.
The plate can be used to crystallize any crystallisable molecule such as a protein or some organic compounds. The volume of the crystallization media to be used with the plate of the present invention will vary, but generally will be of at least 1 μL, more preferably about 5 to about 500 μL, and most preferably 10 μL of said crystallization media. Preferably, the crystallization media is contained in a crystallization media reservoir to the substrate well.
In accordance with the present invention, there is also provided a method for optimizing crystallization conditions for a substrate comprising the step of adding said substrate into each well of a plate as defined above.
The method may additionally further comprise adding a hit solution for said substrate in each well before or after adding said substrate in each well.
Further in accordance with the present invention, there is provided a method for optimizing crystallization conditions for a substrate comprising the step of contacting said substrate with a hit solution for said substrate, and said crystallization media into each well of a plate as defined above.
Still in accordance with the present invention, there is provided a method for optimizing crystallization of a substrate comprising:
In a further embodiment of the present invention, there is also provided a method for optimizing crystallization conditions for a substrate comprising:
Applicant has found that by using the above-mentioned plate or methods, it is possible to rapidly optimize the crystallization conditions for a given substrate. Moreover, when using such a plate or methods, it is possible to rapidly obtain considerable amount of information concerning optimal conditions for a given substrate. This plate or these methods permit to directly use an initial hit solution, hereby improving the reproducibility, and straightforward analysis. Moreover, this plate or these methods permit a wider coverage of the crystallization space. It is also possible to carry out a direct testing of concentration, pH variation and additives effect on crystallization.
In the plate or methods of the invention, the parameters can be selected from the group consisting of a buffer, pH of the crystallization media, salt, concentration of the salt, temperature of the crystallization media, additive, concentration of the additive, ligand (or co-crystallization compound), concentration of the ligand, alcohol, concentration of the alcohol, polymer, concentration of the polymer.
The method and apparatus of the invention are useful for optimizing crystallization conditions of substrates such as proteins.
Further features and advantages of the invention will become more readily apparent from the following description of preferred embodiments as illustrated by way of examples in the appended drawings wherein:
In accordance with one embodiment of the invention, there is presented herewith a strategy which takes advantage of a closer connection between 2 elements of a successful crystal growth experiment: Initial screening and Optimization. The proposed strategy combines a variation in the original initial screening and a subtle change in its analysis.
Initial screening is combined with optimization to minimize time and protein use, while maximizing success. This is however not done easily since one of the problems is the biased and incomplete analysis of the initial screen results. it is biased since i) classification and optimization is only performed around observable crystal forms, and ii) all drops not showing a crystal form are scored and kept aside.
Usually, initial screens gives “Initial hits”. If lucky, these hits will contain high resolution crystals and the protein structure will be solved easily. But it is rarely the case. In most case, one can expect to obtain:
If “Missed-Hits” are not paid attention to, the “Best-hit” may be missed altogether simply because something else than a crystal form was seen in the initial screening.
The present invention thus allows maximizing success by improving initial screening results analysis to select the optimization technique.
Presented herein in accordance with the present invention is a new method where the selection of the crystallization solution and experiment scoring in initial screening strategy are modified to get more information on protein solubility behavior. An analysis of the results, paying close attention to those “Missed-Hits”, guides the crystallographer toward the proper optimization strategies to use next. Essentially the method comprises the steps of:
Using this crystallization strategy, less protein is required, which allows for more analysis, less time is also required to obtain a best hit from a protein in solution to an X-ray quality crystal, and consequently, money is saved by using less protein and taking less time.
The integration of an initial screening with an optimization step as in the method of the present invention is illustrated in
In accordance with a preferred embodiment of the present invention, there is also provided a new plate was developed to facilitate and accelerate optimization set up while respecting experimental constraints. This new plate will be called, hereinafter, the Optimizer plate. Such a plate comprises:
It has been found that by simply adding 90 μL of an initial hit solution (following an initial screen—see
The principal advantages of this Pre-Filled optimization plate are:
It has been shown in table 2, that when using a pre-filled optimizer plate, clear improvement of crystalline form quality can be observed, more suitable crystals are obtained, and different crystal forms for the same protein can be also obtained. The set-up is much simpler and faster and the “time-to-crystal” is reduced.
Of course, one skilled in the art will appreciate that the method and Optimizer plate of the present invention can make use of more different conditions, so as to fill up a plate.
The Mini-grid optimization approach (see
From table 3, it can be seen that, depending on the protein to crystallize and the initial condition, different optimization components show different influences, demonstrating the importance of a wider sampling of crystallization space in optimization strategies.
Table 5 provides for a summary of the results obtained with the conditions of Table 4 and illustrated in
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
Table 6 below lists the current content of one of the plate design for optimization of crystallization designed by the Applicant. Of course numerous other modifications could be made, be the example is only being given for illustrative purpose. To be noted that two negative controls have been introduced to confirms results obtained, i.e. well no. 1 and well no. 13. Well no. 1 has been left empty to verify the reproducibility of the assay and well no. 13 was filled with equal volume (compared to the other wells) of water to verify the effects of dilution on the initial parameters. The controls have never been used in such an assay as in initial screening, there is no incentive to leave blank well. Thus one skilled in the art would not be led to create a plate as the one in Table 6, with the two control wells.
In this experiment, pre-filled optimizer plate (Greiner 3 well format) was used to optimize co-crystallization condition between a protein and 3 different compounds. Optimized crystallization condition of the native protein was added and mixed in each well of the pre-filled plate.
Each chemical compound having its own characteristics can interfere with the stability/interaction of the crystallization process, possibly preventing the crystallization in the initial condition. The Optimizer plate allows creating small grids around a successful crystallization condition of a protein and finding a proper condition for co-crystallization between the protein and chemical compounds. Shown in
An initial crystallization hit consisting of very thin, needle crystals, not usable for X-ray diffraction was obtained with The Classics Suite. No improvement was achieved when using usual optimization strategy. As a complementary approach, 90 μL of the initial hit solution (unknown protein to be crystallized pursuant to a research contract made by the Applicant—the identity and nature of the protein being kept secret to the Applicant) was added and mixed in each well of the optimizer multi-well plate (Corning conical flat bottom format) and used for optimization. Two very distinct and large protein crystals grown (see
As demonstrated in the above examples, using the crystallization plate of the invention, it has been possible to successfully optimized crystallization conditions for 5 commercially available proteins. Starting with needles, microcrystals and even granular precipitates, suitable crystals have been obtained. In the two above-mentioned case studies, The optimizer plate was key in the production of co-crystals between a protein and 3 different ligands and well defined 3D crystals (2.8 Å on home source) of an important protein target. For each of the case study, results were obtained in a single microplate, prepared in minutes. Every experiment led to a variety of results from clear drop to heavy precipitate, showing the influence of the optimization solution mix on the protein solubility. The use of a variety of salts as optimizers highlights the differences between the cation (sodium, potassium, ammonium, magnesium, calcium and lithium) and the anion (formate, malonate, chloride, acetate, fluoride, nitrate, thiocyanate, etc) part of salts. Other Optimizer plates using Pegs, organics and other chemicals as co-precipitant can also be used.
This new pre-filled optimizer plate represents a promising alternative to a standard grid approach when performing optimization. It is easier and faster to setup and bring a lot of information on effect of salt concentration, buffers, and additives on crystallization of a particular protein. Effective 96 optimization conditions can be prepared in less than 10 minutes.
The optimization strategy described herein can be applied as soon as crystal forms appear in a drop. It is a faster and easier method than those now in existence. The simple addition of someone's hit condition to each of the 96 chemicals in the pre-filled plate makes this optimization technique rapid and simple. Since the chemical compositions of these micro-plates are so different, the results are actually a 2nd level of screening based on a partly successful 1st level initial screening. By using this simple method, it is now possible to rapidly see if a “mild change” in the chemical environment will be beneficial or not, compare to a very “soft change” brought in by a factorial approach of optimization, as is currently being done.
In a successful crystallization strategic plan, two (2) aspects of crystallization, i.e. an initial screening and an optimization, must be integrated. To maximize the interaction of the two, results from one technique must be easily processed and bring success in the following one. In this case, while working with 6 specific proteins, this interaction between initial screening and optimization was tested on 54 different crystallization results. Once the optimization technique was selected, major improvement was seen in 85% of the cases (46/54). By combining an initial screening plan (large chemical variety with 2 concentration of precipitants) and a solid optimization procedure like the Optimizer plate, it is now possible to react rapidly during crystal growth and get the sought after success, i.e. diffraction-quality crystals.
Of course, one skilled in the art will readily appreciate that the present invention as now disclosed can also be used as a transfer plate, and not only a crystallization plate. For example, plates containing in each well sufficient optimizing solutions (crystallization media) for a number of assays could be used and sold. instead of 10 μl be put in each well, a plate that would have 250 μl per well could thus be used for 10 assays (assuming there is no loss or evaporation of the media). Furthermore, the person skilled in the art will appreciate that correction of concentration of the reagents (for example the hit solution) may be desired.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA05/01118 | 7/15/2005 | WO | 00 | 11/24/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60588359 | Jul 2004 | US |
