Strategies for Protein Cryocrystallography

Cryoprotection is the final step before flash-cooling, during which crystals can be improved or damaged and data quality maximized. A well thought-out cryoprotection requires optimized composition of suitable components and an appropriate soak period. Crystallization methodology has been extensively studied, but not cryoprotection. Cryoprotectant selection remains a trial and error exercise where the first combination that “works” is accepted. The approach presented here consists in a formulation of a few mixed compounds to speed-up crystal preparation for synchrotron data collection. A good cryoprotectant solution needs to stabilize the crystalline state and prevent ice formation during flash-cooling, its composition can differ widely from the crystallization precipitant since it is not required to induce nucleation, establish an equilibrium between the protein crystal and the solution or permit crystal growth. The strategy presented here differs from the general practice that involves a single anti-icing chemical, often glycerol, a molecule able to enhance protein solubility. The multicomponent mixtures selected through an evolutive process neither increase nor decrease protein solubility but provide extended stabilization during cryoprotection to allow longer soak periods without crystal cracking or dissolving.