Today the determination of successful crystallization conditions for a particular macromolecule remains a highly empirical process. Sparse-matrix and grid-screening procedures are rapid and economical means to determine preliminary crystallization conditions. During optimization the variable set (pH, precipitant type and precipitant concentration) utilized in these procedures is screened in an attempt to determine appropriate conditions for the nucleation and growth of single crystals suitable for X-ray diffraction analysis. Unfortunately, in many cases this strategy will not produce single crystals suitable for X-ray diffraction analysis. We have explored, in an empirical sense, other tools for use during optimization. First, a new screening protocol is evaluated which employs less classical precipitating agents. Second, a set of 24 electrostatic crosslinking agents are evaluated for their ability to promote crystallization. Third, a panel of more than 30 detergents are evaluated for their ability to prevent sample aggregation and influence crystal growth.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

An alternative approach to promoting the crystallization of proteins is to enhance intermolecular contacts between macromolecules or to eliminate intermolecular interactions or interactions with solvent that might inhibit crystallization. Site-specific mutations have been employed, as have truncations by genetic or proteolytic means. There are, however, significant problems. Because the structure of the target macromolecule is unknown, there may be no good basis for the design of mutants or truncations. In addition, the approach requires that the protein be produced by recombinant DNA technology, which is frequently not the case. We have attempted to address these issues by initiating experiments based on two ideas. The first is that a wide variety of conventional small molecules might be systematically introduced into mother liquors during crystallization screening. By incorporation into the crystal lattice, the additional intermolecular interactions that the small molecules provide might enhance crystal nucleation and growth. A second approach that we are pursuing is the chemical modification of various amino acid side chains using traditional protein chemistry. We believe that in some cases chemically modified proteins might be induced to crystallize or crystallize better than the native protein.

Recently, the hypothesis was advanced that protein crystallization could be driven by the inclusion of small molecules rich in hydrogen‐bonding, hydrophobic and electrostatic bonding possibilities. Conventional organic and biologically active molecules would promote lattice formation by their mediation of intermolecular interactions in crystals. The results of an extensive series of crystallization experiments strongly supported the idea. Here, difference Fourier X‐ray diffraction analyses of nine crystals grown in the experiments are presented, which convincingly demonstrate the validity of the hypothesis and illustrate some of the ways in which small molecules can participate in lattice interactions.

Procedures are described for the crystallization of proteins, nucleic acids and viruses in a silica-gel matrix using otherwise standard reagents and conditions. Methods are given based on both vapor diffusion in a sitting drop and liquid-liquid diffusion. Using a variety of macromolecules our results suggest that the gel matrix suppresses nucleation, reduces the rate of growth, and generally leads to larger, higher-quality crystals of enhanced stability. Presumably these effects arise from the decreased mobility of the macromolecules and their flux at the crystal surface during growth.

We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, noncovalent cross-links in crystals, and thereby promote lattice formation. Earlier experiments provided encouraging results that suggested further research was warranted (Larson,

PDB Reference: pig heart citrate synthase, 3enj, r3enjsf.Pig heart citrate synthase was crystallized from a small-molecule cocktail containing cystamine dihydrochloride, aspartame and benzamidine hydrochloride. The structure was refined to an R factor of 0.179 (R free = 0.222) using synchrotron data to a resolution of 1.78 Å . The model includes the full-length protein, a chloride ion, a sulfate ion, 305 water molecules and an unexpected moiety attached through a disulfide linkage to Cys184, which was modeled as a half-cystamine molecule generated by disulfide exchange with the cystamine in the small-molecule cocktail.

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