Section 4.1.2.2. Batch crystallizations

Batch methods are the simplest techniques used to produce crystals of macromolecules. They require no more than just mixing the macromolecular solution with crystallizing agents (usually called precipitants) until supersaturation is reached (Fig. 4.1.2.1a). Batch crystallization has been used to grow crystals from samples of a millilitre and more (McPherson, 1982), to microdroplets of a few µl (Bott et al., 1982), to even smaller samples in the µl range in capillaries (Luft et al., 1999a). Because one begins at high supersaturation, nucleation is often excessive. Large crystals, however, can be obtained when the degree of supersaturation is near the metastable region of the crystal–solution phase diagram.

An automated system for microbatch crystallization and screening permits one to investigate samples of less than 2 µl (Chayen et al., 1990). Reproducibility is guaranteed because samples are dispensed and incubated under oil, thus preventing evaporation and uncontrolled concentration changes of the components in the microdroplets. The method was subsequently adapted for crystallizing proteins in drops suspended between two oil layers (Chayen, 1996; Lorber & Giegé, 1996). Large drops (up to 100 µl) can be deployed, and direct observation of the crystallization process is possible (Lorber & Giegé, 1996). The absence of contacts between the mother liquor and any solid surfaces yields a reduced number of nucleation sites and larger crystals. Batch crystallization can also be conducted under high pressure (Lorber et al., 1996) and has also been adapted for crystallizations on thermal gradients with samples of ∼7 µl accommodated in micropipettes (Luft et al., 1999b). This latter method allows rapid screening to delineate optimal temperatures for crystallization and also frequently yields crystals of sufficient quality for diffraction analysis.

Batch methods are well suited for crystallizations based on thermonucleation. This can be done readily by transferring crystallization vessels from one thermostated cabinet to another maintained at a higher or lower temperature, depending on whether the protein has normal or retrograde solubility. In more elaborate methods, the temperature of individual crystallization cells is regulated by Peltier devices (Lorber & Giegé, 1992). Local temperature changes can also be created by thermonucleators (DeMattei & Feigelson, 1992) or in temperature-gradient cells (DeMattei & Feigelson, 1993). A variation of classical batch crystallization is the sequential extraction procedure (Jakoby, 1971), based on the property that the solubility of many proteins in highly concentrated salt solutions exhibits significant (but shallow) temperature dependence.