Cryosolution thermal contraction is found to have an impact on cryocooled protein and unit-cell volumes and conformations. In some cases, its adjustment can produce higher quality diffraction data.
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 nitrogen 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.
nanoparticle-protein conjugates are not easily crystallized. Previous work from our group suggests that the GB3 protein remains globular when adsorbed to gold nanoparticles (AuNPs), but it is unclear whether the tertiary structure is retained. Here, we apply several novel NMR-based approaches to probe the structure and orientation of GB3 bound to AuNPs. We have developed a method for monitoring hydrogen-deuterium exchange (HDX) on the AuNP surface, and we find that HDX rates of surface-bound GB3 are highly correlated with GB3 in solution. Overall, rates are approximately 20 times slower for the adsorbed protein, suggesting that GB3 is stabilized and largely retains its native structure on the surface. Methyl labeling of lysine residues suggests that the orientation of GB3 is fixed on the AuNP, with the helical face exposed to solution. Using differential isotopic labeling, we have determined that adsorbed GB3 molecules do not readily exchange with GB3 in solution, and any exchange that happens occurs on a timescale much longer than 18 hr. These experiments provide strong structural evidence that GB3 adopts a stable, native-like fold and orientation on the AuNP surface, and they open the door for future investigations of protein structure on surfaces.
Macromolecular structure determination via diffraction is commonly carried out at cryogenic temperature. Crystals are typically cooled via plunging into liquid cryogens or placing directly into a cold gas stream. Here we compare these two approaches using two different crystal forms of thermolysin. We find that fast plunge cooling of ~300 μm crystals into liquid nitrogen yields higher mosaicities than gas stream cooling using the vial mounting approach (Farley et al., 2014). In some cases low mosaicities can also be achieved by plunging slowly through the cold gas layer above the liquid nitrogen. The observed effects are more pronounced for a cryosolution of DMF (10% contraction) than for a cryosolution of D-xylose (3% contraction). The results are consistent with a model in which non-homogeneous cooling-induced strain is amplified by faster cooling of greater contracting materials (Kriminski et al., 2003).
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