Chapter 3: Annealing Macromolecular Crystals
B. Leif Hanson & Gerard J. Bunick
The process of crystal annealing has been used to improve the quality of diffraction from crystals that would otherwise be discarded for displaying unsatisfactory diffraction after flash cooling. Although techniques and protocols vary, macromolecular crystals are annealed by warming the flash-cooled crystal, then flash cooling it again. To apply macromolecular crystal annealing, a flash-cooled crystal displaying unacceptably high mosaicity or diffraction from ice is removed from the goniometer and immediately placed in cryoprotectant buffer. The crystal is incubated in the buffer at either room temperature or the temperature at which the crystal was grown. After about 3 min, the crystal is remounted in the loop and flash cooled. In situ annealing techniques, where the cold stream is diverted and the crystal allowed to warm on the loop prior to flash cooling, are variations of annealing that appears to work best when large solvent channels are not present in the crystal lattice or the solvent content of the crystal is relatively low.