W0200

The Role of Water Transport in Cryo-Annealing. Douglas H. Juers and Brian W. Matthews, HHMI, Institute of Molecular Biology and Dept. of Physics, Univ. of Oregon, Eugene, OR 97405.

Cryocooling of macromolecular crystals for X-ray data collection has become standard practice in recent years. However, in many instances, cryocooling is far from routine and often great labor is required to arrive at appropriate cryocooling conditions. In some cases, the diffraction quality of cryocooled crystals can be improved with annealing procedures which involve repeated cycles of cryocooling and melting. We have studied the reversibility of cryocooling in crystals of E. coli β-galactosidase under different humidity conditions. With repeated cycles of cooling (with a standard cold flow at ~100 K) and in-situ melting by blocking the cold stream, the crystal either absorbs or desorbs water, depending on the ambient humidity and the duration of the melt cycle. If the initial cryoprotectant concentration is greater than optimal, the absorption of water during the melt cycle can dramatically improve diffraction quality (e.g. from 4 Å to 2 Å).

This suggests that at least part of the mechanism of annealing is to allow the crystal to either absorb or desorb water, effectively adjusting the cryoprotectant concentration. Density measurements suggest that the optimal cryoprotectant concentration may be that which allows the contraction of the bulk solvent to compensate for the contraction of the macromolecular lattice. These experiments demonstrate that humidity control can be helpful in identifying optimal cryocooling conditions.