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Certain chemical compounds or small molecules may have dramatic effects on the success with which individual proteins crystallize. While additives, as they are often called, can be decisive in macromolecular crystallization, their greater use has suffered from lack of any compelling, rational basis for their inclusion in mother liquors. The most commonly useful class of additives, and the only class of which we have any real understanding, are those which may, for physiological reasons, be bound by the protein with consequent favorable changes in its physical–chemical properties or conformation. These include such things as coenzymes and prosthetic groups, inhibitors, enzymatic products, ions, and other effector molecules. Often the liganded form is structurally defined and stable, while the unliganded form is not, and often the former will crystallize when the latter will not.

Numerous cases have, however, been reported where small, and sometimes large molecules were observed to make crucial interactions between macromolecules in the crystal that either helped guide or secure formation of the lattice. Such small molecules sometimes had a physiological basis for their unexpected presence, but frequently not. They simply provided essential or at least helpful crosslinks within the crystal. A casual poll of crystallographic colleagues provided a list of 50 or so molecules identified (or at least believed to be so), which had been unintentionally incorporated into various protein crystals. There are undoubtedly many more.

Therefore, based on a hypothesis that various small molecules might establish stabilizing, intermolecular, non covalent crosslinks in protein crystals and thereby promote lattice formation, Dr. Alexander McPherson and his colleagues at the University of California at Irvine (UC Irvine) have assessed the impact of 200 chemicals on the propensity of 81 different proteins and viruses to crystallize. The experiments were comprised of 18,240 vapor diffusion trials. A salient feature of the experiments was that, aside from the inclusion of the reagent mixes, only two fundamental crystallization conditions were used, 30% PEG 3350, and 50% Tacsimate™ (Hampton Research, Aliso Viejo, CA) at pH 7.  The latter, a new precipitant composed of seven different pH neutralized organic acid salts, has seen increasing success not only because it produces high ionic strengths, but almost certainly because it provides a diverse set of molecules that can form hydrogen bonding or electrostatic, reversible crosslinks between proteins in the crystal lattice.

Overall, 65 proteins (85%) were crystallized. Most significant was that 35 of the 65 (54%) crystallized only in the presence of one or more reagent mixes, but not in control samples lacking any additives. Among the most promising types of reagent mixes were those composed of polyvalent, charged groups, such as di and tri carboxylic acids, diamino compounds, molecules bearing one or more sulfonyl or phosphate groups, and a broad range of common biochemicals, coenzymes, biological effectors, and ligands.  Many crystals obtained in these experiments were microcrystals, but a significant number, as evidenced by the pictures on this page, were immediately suitable for X-ray diffraction analysis. Also, in many cases, different crystal forms appeared depending on the reagents used.

The collaboration between UC Irvine (developing more effective crystallization cocktails) and the Hauptman-Woodward Institute (HWI) (high-throughput crystallization methods) creates an opportunity for a group of "silver bullet" cocktails to be integrated into the 1536 screening solutions used by the high-throughput crystallization laboratory at HWI.


McPherson, A. & Cudney, R. (2006). Searching for silver bullets: an alternative strategy for crystallizing macromolecules. J. Struct. Biol. 156, 387–406.  [PubMed]

Larson, S. B., Day, J. S., Cudney, R. & McPherson, A. (2007). A novel strategy for the crystallization of proteins: X-ray diffraction validation. Acta Crystallogr. D63, 310–318. [PubMed]