Section 4.1.4.1. Rules and general principles

The first concern is to obtain a macromolecular sample of highest quality; second, to collate all biochemical and biophysical features characterizing the macromolecule in order to design the best crystallization strategy; and finally, to establish precise protocols that ensure the reproducibility of experiments. It is also important to clean and sterilize by filtration (over 0.22 µm porosity membranes) all solutions in contact with pure macromolecules to remove dust and other solid particles, and to avoid contamination by microbes. Inclusion of sodium azide in crystallizing solutions may discourage invasive bacteria and fungi. In vapour-diffusion assays, such contamination can be prevented by simply placing a small grain of thymol in the reservoir. Thymol, however, can occasionally have specific effects on crystal growth (Chayen et al., 1989) and thus may serve as an additive in screenings as well.

Crystallization requires bringing the macromolecule to a supersaturated state that favours nucleation. Use of phase diagrams may be important for this purpose (Haas & Drenth, 1998; Sauter, Lorber et al., 1999). If solubilities or phase diagrams are unavailable, it is nevertheless important to understand the correlation between solubility and the way supersaturation is reached in the different crystallization methods (see Fig. 4.1.2.1). In dialysis, the macromolecule concentration remains constant during equilibration. The initial concentration of the crystallizing agent in the exterior solution leaves the macromolecule in an undersaturated state. With increasing concentration of the agent in the exterior solution, a state of supersaturation can be attained, leading to crystallization or precipitation. In a vapour-diffusion experiment, where the concentration of crystallizing agent in the reservoir exceeds that in the drop, the macromolecule will begin to concentrate from an undersaturated to a supersaturated state, with both macromolecule and crystallizing-agent concentrations increasing. Crystals appear in the metastable region. For crystals that appear first, the trajectory of equilibration is complex and the remaining concentration of macromolecule in solution will converge towards a point located on the solubility curve. In batch crystallization using a closed vessel, three situations can occur: if the concentration of the macromolecule is undersaturated, crystallization never occurs (unless another parameter such as temperature is varied); if it belongs to the supersaturated region between solubility and precipitation curves, crystals can grow until the remaining concentration of the macromolecule in solution equals its solubility; if supersaturation is too high, the macromolecule precipitates immediately, although in some cases, crystals can grow from precipitates by Ostwald ripening (Ng et al., 1996).