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.

[1]  F. Travers,et al.  Dielectric constants of alcoholic-water mixtures at low temperature , 1970 .

[2]  P. Douzou.  Aqueous-organic solutions of enzymes at sub-zero temperatures , 1971 .

[3]  D. Tsernoglou,et al.  Cytoplasmic malate dehydrogenase--heavy atom derivatives and low resolution structure. , 1972, Journal of molecular biology.

[4]  Walter Hoppe,et al.  Freezing of myoglobin crystals at high pressure , 1973 .

[5]  P. Douzou.  Le contrôle thermique des réactions enzymatiques , 1971 .

[6]  Gösta. Åkerlöf,et al.  DIELECTRIC CONSTANTS OF SOME ORGANIC SOLVENT-WATER MIXTURES AT VARIOUS TEMPERATURES , 1932 .

[7]  G. Petsko,et al.  Protein crystallography at sub-zero temperatures: lysozyme-substrate complexes in cooled mixed solvents. , 1975, Journal of molecular biology.

[8]  N. Allewell,et al.  Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S. , 1967, Journal of molecular biology.

[9]  M. Rossmann,et al.  Crystallographic studies on lactate dehydrogenase at –75°C , 1970 .

[10]  D. Phillips.  On the design of single crystal diffractometers to measure a number of reflections simultaneously , 1964 .

[11]  G. Petsko,et al.  A low‐temperature device for protein crystallography , 1973 .

[12]  P. Douzou,et al.  Ionic strength and protonic activity of supercooled solutions used in experiments with enzyme systems. , 1973, The Journal of biological chemistry.