One of the obstacles to structure determination of macromolecules by X-ray crystallography is radiation-induced crystal decay. Crystal decay appears to be a result of X-ray radiation and thermal damage to the macromolecule and, subsequently, to the lattice. This damage hinders uninterrupted data collection and introduces experimental errors to the measurements. In the past the only way to overcome this obstacle was to collect data from several crystals, switching to the next when the previous one decayed beyond usable levels. This strategy suffers from problems of scaling datasets from crystal to crystal. Radiation and thermally induced crystal decay can be slowed and even stopped entirely by collecting data from cooled crystals.
To flash-cool, in this context, means to take a specimen from non-cryo-conditions to cryo-conditions rapidly. Plunging a protein crystal into cryo-temperature causes the aqueous solution, in and around the crystal, to freeze amorphously, like glass, in a process known as vitrification (Mayer, 1985; Dubochet and Schultz, 1988). In principle, vitrification should not change the protein crystal in terms of lattice integrity and the solute structure. In practice, flash-cooling of a crystal can affect the lattice (Low et al., 1966; Singh et al., 1980; Kellenberger, 1987). As a consequence the crystal may exhibit a change in the degree of order of the lattice (the mosaic spread). In most cases, the mosaicity will increase upon flash-cooling, but there are cases where the opposite effect is observed (Zalonga and Sarma, 1974; Young et al., 1990). This behavior is greatly influenced by the solute composition. To increase the rate of success of flash-cooling, an additive can be introduced to the mother liquor. The term "cryo-protectant" refers to the chemical or chemicals that permits the cooling of a protein crystal with little or no increase in its mosaicity (Haas and Rossmann, 1970; Petsko, 1975; Casico et al., 1984; Dewan and Tilton, 1987; Dubochet and Schultz, 1988; Hope et al., 1989). Those chemicals, which have so far been members of the alcohol, sugar or sulfoxide families, serve to slow the nucleation of ice, raise the viscosity of the solution as it cools (and thus raise the glass transition temperature) and break the propagation of ice formation in the mother liquor. Those additives are added in amounts ranging from as low as 2% to as high as 30% by volume.
Other parameters that affect the freezing process are the characteristics of the crystal itself such as size and shape, mechanical stability and density. When subjected to flash-cooling, the crystal is suspended on a support surrounded by the aqueous solvent. The nature of this support and the amount of solvent surrounding the crystal is another important parameter in the freezing process. In the design of the support the mechanical strength of the crystal should be considered, as well as minimization of the amount of mother liquor surrounding it.
Once a crystal has been frozen, it can no longer be taken out of cryo-conditions without almost total loss of diffraction. As the solvent warms up, slowly or rapidly, it will go through several phase changes and ice will form within and around the crystal, causing the breakdown of the lattice. Thus, storage of flash-frozen crystals at cryo-temperatures is imperative.