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Crystallization
The determination of the three-dimensional structures of proteins by X-ray crystallography requires the availability of large, well-ordered crystals. These are difficult to obtain. The crystallization of any given protein requires long painstaking work, including much trial and error in the choice of environmental conditions (pH, temperature) and crystallizing agents.
The basic requirement is the availability of the protein in very pure form, free of all other molecules, such as traces of nucleic acids, in a single form (absence of isoforms), in a solution that is free of any dust particles. If liganded to any coenzymes, cofactors, or effectors, all molecules must be equally liganded; all molecules must be in the native state, and there must be no partial oligomer formation.
The basic approach is to prepare a protein solution at high concentration (10 to 100 mg/ml) in a crystallizing solvent at conditions at which the solution is close to saturation. Then the solution is gradually brought to supersaturation by, eg, slow variation in pH or temperature. All changes must be brought about slowly and the solution not subjected to shocks, such as mixing. This allows the formation of crystal nuclei. Amorphous aggregates must be totally avoided. Once nuclei are formed, the system is induced to crystallize further by the addition of protein to these nuclei, avoiding the formation of new ones, since the aim is to form large crystals. See the specialized literature for further details.
Typical crystallizing agents are (1) salts (NH4)2SO4, Na2SO4, Na citrate, MgSO4; (2) organic molecules [2-methyl-2,4-pentanediol (MPD), ethanol, isoproponal, acetone, dioxane, 1,3-propanediol]; (3) polyethylene glycols (molecular weight between 2,000 and 20,000).
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