Chemical apparatus and process disinfecting – deodorizing – preser – Physical type apparatus – Crystallizer
Reexamination Certificate
1999-06-25
2001-07-31
Powell, William (Department: 1765)
Chemical apparatus and process disinfecting, deodorizing, preser
Physical type apparatus
Crystallizer
C436S004000
Reexamination Certificate
active
06267935
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to solutions that are useful in the crystallization of molecules, especially macromolecules such as proteins.
BACKGROUND OF THE INVENTION
Macromolecular X-ray crystallography is an essential tool in modern drug discovery and molecular biology. Using X-ray crystallographic techniques, the three-dimensional structures of biological macromolecules, such as proteins, nucleic acids, and their various complexes, can be determined at practically atomic-level resolution from X-ray diffraction data.
One of the first and most important steps in the X-ray crystal structure determination of a target macromolecule is to grow large, well-diffracting crystals of the macromolecule. As the techniques for collecting and analyzing X-ray diffraction data have become 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. The resulting drop of liquid (containing macromolecule and dilute crystallization solution) is sealed in a chamber with a much larger reservoir volume of the crystallization solution. The drop is kept separate from the reservoir of crystallization solvent either by hanging the drop from a glass cover slip or by sitting the drop on a pedestal above the level of the solvent in the reservoir. Over time, the crystallization drop and the reservoir solutions 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.
The process of growing biological macromolecule crystals remains, however, a highly empirical process. Macromolecular crystallization is dependent on a host of experimental parameters, including; pH, temperature, the concentration of salts in the crystallization drop, the concentration of the macromolecule to be crystallized, and the concentration of the precipitating agent (of which there are hundreds). In particular, the choice of solute conditions in which to grow crystals continues to be a matter for empirical determination. Consequently, the ability to rapidly and easily generate many crystallization trials is important in determining the ideal conditions for crystallization. Thus, there is a need for sets of preformulated crystallization solutions that can be used to rapidly and easily generate many crystallization trials.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides solutions (hereinafter referred to as crystallization solutions) useful for crystallizing proteins and other molecules, especially macromolecules. In a presently preferred embodiment, the crystallization solutions of the invention are combined as four crystallization solution sets (identified herein as Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV). Each of the four crystallization solution sets includes forty eight different crystallization solutions. Each individual crystallization solution includes a precipitant and a buffer, and optionally includes at least one additive as set forth more fully herein. The compositions of the individual solutions that constitute Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV are set forth in Table I, Table II, Table III and Table IV, respectively. Thus, in one aspect, the present invention is directed to sets of crystallization solutions including the solutions of one or more of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV.
In another embodiment, the present invention provides subsets of the crystallization solutions of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV which are useful to determine crystallization conditions for a wide variety of molecules, especially biological macromolecules. While any subset of crystallization solutions of Crystallization Solution Set L Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV can be utilized, presently preferred crystallization solution subsets are: subset I, solution numbers 1, 9, 10 and 28 of Crystallization Solution Set I; subset II, solution numbers 1, 8, 14, 26, 30 and 34 of Crystallization Solution Set II; subset III, solution numbers 2, 11, 18, 25, 26, 28, 29, 31, 38, 41 and 46 of Crystallization Solution Set III; and subset IV, solution numbers 2, 3, 5, 6, 7, 8, 17, 22, 24, 32, 34, 37, 43 and 48 of Crystallization Solution Set IV. Thus, crystallization solution subsets preferably include at least one of subset I, subset II, subset III and subset IV. As described more fully herein, the foregoing, presently preferred subsets have been successfully used to crystallize several proteins, including proteins considered recalcitrant to crystallization.
In another aspect, the present invention provides kits including a plurality of crystallization solutions of the present invention and at least one crystallization plate that preferably includes a plurality of reservoirs. Preferably the crystallization solutions of the crystallization solution set(s) are disposed within the reservoirs of the crystallization plates. The presently preferred crystallization plates are disclosed in copending U.S. patent application Ser. No. 09/150,629, issued on Mar. 21, 2000 as U.S. Pat. No. 6,039,804 incorporated herein by reference. In a presently preferred embodiment, the present invention provides kits including at least one crystallization plate and a set of crystallization solutions selected from the group of sets consisting of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV. In another presently preferred embodiment, the present invention provides kits including at least one crystallization plate and including a subset of crystallization solutions selected from the group of subsets consisting of: subset I, solution numbers 1, 9, 10 and 28 of Crystallization Solution Set I; subset II, solution numbers 1, 8, 14, 26, 30 and 34 of Crystallization Solution Set II; subset III, solution numbers 2, 11, 18, 25, 26, 28, 29, 31, 38, 41 and 46 of Crystallization Solution Set III; and subset IV, solution numbers 2, 3, 5, 6, 7, 8, 17, 22, 24, 32, 34, 37, 43 and 48 of Crystallization Solution Set IV.
Thus, the present invention provides solutions and kits that permit a large number of crystallization conditions to be easily and simultaneously tested in order to identify crystallization conditions under which a target molecule, especially a biological macromolecule such as a protein, can be crystallized. As discussed more fully herein, the crystallization solutions of the present invention have been successfully used to crystallize proteins regarded as recalcitrant to crystallization.
REFERENCES:
patent: 5130105 (1992-07-01), Carter et al.
patent: 6093573 (2000-07-01), Beamer et al.
Screening Kits: Product Information Sheets, 2 pp., Downloaded Sep. 21, 1999 from Hampton Research Website www.hamptonresearch.com/211x.html.
Hampton Research Solutions for Crystal Growth, Crystal Screen Reagent Formulations, Listing, 3 pp., Downloaded Sep. 21, 1999 from Hampton Research Website www.hamptonresearch.com/2110rw.html.
Hampton Research Solutions for Crystal Growth, Crystal Screen 2 Reagent Formulations, Listing, 3 pp., Downloaded Sep. 21, 1999 from Hampton Research Website ww.hamptonresearch.com/2112rw.html.
Hampton Research Solutions for Crystal Growth, Crystal Screen Cryo Reagent Formulations, Listing, 3 pp., Downloaded Sep. 21, 1999 from Hampton Research Websit
Hol Wim G. J.
Kim Hidong
Sarfaty Steven H.
Stewart Lansing J.
Christensen O'Connor Johnson & Kindness PLLC
Powell William
University of Washington
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