Cryocrystallography of macromolecules: practice and optimization

Techniques for flash-cooling protein crystals to around 100K (-173 degrees C) for data collection have developed enormously in the last decade, to the extent that cryocrystallography is now standard practice. The main advantage of these methods is the vastly reduced rate of radiation damage to protein crystals in the X-ray beam at cryogenic temperatures over room temperature, extending their lifetimes so that complete datasets can be collected from a single crystal. The practical application of the techniques has become somewhat anecdotal and rather fixed within individual laboratories. This chapter gives step-by-step guidelines for flash-cooling crystals and some of the rationale for the recommended procedures. Optimization of the entire cryoprotocol can give substantial improvements to both the resolution and quality of the data, often resulting in more straight-forward structure solution and subsequent model refinement. Attention to seemingly insignificant details can have a real impact on the usefulness of the final dataset, and are thus worth addressing.