Protein crystallography at sub-zero temperatures: Cryo-protective mother liquors for protein crystals

Abstract

A procedure has been developed for maintaining the integrity of protein crystals at sub-zero temperatures. It involves the replacement of the normal crystal mother liquor with salt-free aqueous/organic liquids of low freezing point. Detailed knowledge of the physical chemical properties of these mixed solvents permits conditions to be chosen that maximize the similarity between their microenvironments and that provided by the normal mother liquor. Since the mixed solvents remain fluid at very low temperatures, it is possible to diffuse substrates into the crystals in the cold. Results have been obtained for a dozen different crystalline proteins, demonstrating that the crystals diffract to high resolution after repeated cooling to below − 70 °C. At low temperatures radiation damage to all the crystals is negligible, and for two proteins there is an improvement in the intensities of high resolution reflections. The technique requires no special equipment, does not change unit cell dimensions, and does not demand cross-linking the crystals.