THE COOLING RATE DURING SHOCK FREEZING IN MACROMOLECULAR CRYSTALLOGRAPHY T.-Y. Teng and K. Moffat, Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637.
Cryocrystallographic study of macromolecules has grown rapidly in recent years1. Data collection of single protein crystal x-ray diffraction at liquid nitrogen temperatures is now routinely conducted at x-ray labs and synchrotron beamlines. Cryocrystallography greatly reduces radiation damage on the crystals, increases diffraction resolution, simplifies sample handling in the experiment and saves valuable protein crystals when compared with conventional data collection around room temperature, altough slightly increased mosaicity is observed in frozen crystals. But, there always remains doubt about the identity between the structure revealed at cryo-temperatures and the structure at physiological temperatures. Further, the average structure observed at low temperature is an ensemble of series structures frozen at different stages in the shock freeze process2. This is especially important when time-resolved x-ray diffraction studies are interpreted.
To answer the question of how shock freezing proceeds, the cooling rates of different cryoprotectant solutions (sucrose, PEG) with different volumes (0.2 - 0.8 mm3) by different cooling agents (cold nitrogen or helium gas, liquid nitrogen and liquid propane) were measured. The result shows, indeed, macromolecular crystals are not instantaneously frozen to their final stage. Freezing is usually complete within the 1 - 2 second and the cooling speed varies from ~50 to 700 deg./s depending on the volume of the sample, the cooling agent used and the temperature at the time of measurement.
Detailed data will be presented along with some guidelines for shock freezing of protein crystals.
1 Rogers, D. W. (1994). Structure, 2, 1135-1140.
2 Moffat, K. and Henderson, R. (1995) Current Opini. in Structural Biology, 5, 656-663.