Abstract
Cryocooling for macromolecular crystallography is usually performed via plunging the crystal into a liquid cryogen or placing the crystal in a cold gas stream. These two approaches are compared here for the case of nitro-gen cooling. The results show that gas stream cooling, which typically cools the crystal more slowly, yields lower mosaicity and, in some cases, a stronger anomalous signal relative to rapid plunge cooling. During plunging, moving the crystal slowly through the cold gas layer above the liquid surface can produce mosaicity similar to gas stream cooling. Annealing plunge cooled crystals by warming and recooling in the gas stream allows the mosaicity and anomalous signal to recover. For tetragonal thermolysin, the observed effects are less pronounced when the cryosolvent has smaller thermal contraction, under which conditions the protein structures from plunge cooled and gas stream cooled crystals are very similar. Finally, this work also demonstrates that the resolution dependence of the reflecting range is correlated with the cooling method, suggesting it may be a useful tool for discerning whether crystals are cooled too rapidly. The results support previous studies suggesting that slower cooling methods are less deleterious to crystal order, as long as ice formation is prevented and dehydration is limited.
Highlights
Diffraction from crystals can be used to determine both structural and dynamical information about macromolecules
In order to counteract radiation damage, macromolecular crystals are often cryogenically cooled before exposure to X-rays, which slows down the radiation damage by limiting free radical diffusion (Garman & Schneider, 1997)
Additional tests using variations of plunging showed that removal of the cold gas layer above the liquid nitrogen had little effect on the mosaicity, whereas moving the crystal slowly through the cold gas layer yielded slight mosaicity reductions in comparison with normal plunge cooling (Table 1)
Summary
Diffraction from crystals can be used to determine both structural and dynamical information about macromolecules. When macromolecular crystals are exposed to X-ray radiation, they accumulate free radicals that cause degradation of the diffraction data quality over time (Garman & Owen, 2006). Increases in crystal disorder and mosaicity reduce the diffraction limit of the crystal, and make data collection more difficult by requiring larger crystal-todetector distances and smaller oscillations. This is acute for crystals with large unit cells and for Laue diffraction [the method of choice for neutron crystallography (Langan & Greene, 2004)], which produce diffraction patterns with closely spaced Bragg diffraction spots
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