Low Temperature Methods in Biocrystallography That Work. Sean Parkin, L-452, BBRP, Lawrence Livermore National Laboratory, Livermore CA., 94550, USA

High resolution structures of biological macromolecules generally require that data be collected at low temperature. There are a number of reasons for this, but the most important include postponement of radiation damage from x-ray exposure until after data collection, reduced thermal motion and the possibility of conformational changes that effectively reduce disorder. Such studies are typically done using synchrotron radiation, but this is not a necessary criterion. Data of comparable quality can be collected using conventional equipment given a sufficient investment of time and effort.

Successful cooling requires that the crystal be transferred from mother liquor at ambient conditions to near liquid nitrogen temperature without damaging the sample. The aqueous nature of the mother liquor means that ice formation will likely destroy the crystal during the cooling process unless it is removed or modified. In the mounting process, one should have confidence that the crystal is protected from unforeseen temperature fluctuations. Unpredictable transitions between energetically similar conformational states, as observed for example in concanavalin A, should be minimized.

Ideally it should be possible to mount and dismount the crystal (for screening purposes) with the sure knowledge that the crystal never experiences temperatures higher than the cold gas stream, which is typically the warmest part of the low-temperature experiment. The use of properly designed and constructed low-temperature tools is also of paramount importance. When the geometric relationship between the cold stream and the crystal mount is optimized, it is relatively easy to keep a crystal cold and frost free indefinitely.

Simple techniques for crystal handling have been developed that ensure (provided the necessary degree of care is taken) knowledge of the temperature history of a crystal throughout the process of mounting, data collection, dismounting, storage and transport etc. of crystals. These methods will be discussed with examples to illustrate the importance of reproducibility and of maintaining a known crystal environment. Some results of recent atomic resolution structures obtained from standard (albeit modified) laboratory diffraction equipment will also be presented.