New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium.

Cryocrystallography is used today for almost all X-ray diffraction data collection at synchrotron beam lines, with rotating-anode generators, and micro X-ray sources. Despite the widespread use of flash-cooling to place macromolecular crystals in the cryogenic state, its use can ruin crystals, trips to the synchrotron, and sometimes even an entire project. Annealing of macromolecular crystals takes little time, requires no specialized equipment, and can save crystallographic projects that might otherwise end in failure. Annealing should be tried whenever initial flash-cooling causes an unacceptable increase in mosaicity, results in ice rings, fails to provide adequate diffraction quality, or causes a crystal to be positioned awkwardly. Overall, annealing improves the quality of data and overall success rate at synchrotron beam lines. Its use should be considered whenever problems arise with a flash-cooled crystal. Helium is a more efficient cryogen than nitrogen and will deliver lower temperatures. Experiments suggest that when crystals are cooled with He rather than N2, crystals maintain order and high-resolution data are less affected by increased radiation load. Individually or in combination, these two techniques can enhance the success of crystallographic data collection, and their use should be considered essential for high-throughput programs.