This week I presented a paper that investigates the differences between crystallographic datasets collected from crystals at RT (room-temperature) and crystals at CT (cryogenic temperatures). Full paper here.
The cooling of protein crystals to cryogenic temperatures is widely used as a method of reducing radiation damage and enabling collection of whole datasets from a single crystal. In fact, this approach has been so successful that approximately 95% of structures in the PDB have been collected at CT.
However, the main assumption of cryo-cooling is that the “freezing”/cooling process happens quickly enough that it does not disturb the conformational distributions of the protein, and that the RT ensemble is “trapped” when cooled to CT.
Although it is well established that cryo-cooling of the crystal does not distort the overall structure or fold of the protein, this paper investigates some of the more subtle changes that cryo-cooling can introduce, such as the distortion of sidechain conformations or the quenching of dynamic CONTACT networks. These features of proteins could be important for the understanding of phenomena such as binding or allosteric modulation, and so accurate information about the protein is essential. If this information is regulartly lost in the cryo-cooling process, it could be a strong argument for a return to collection at RT where feasible.
By using the RINGER method, the authors find that the sidechain conformations are commonly affected by the cryo-cooling process: the conformers present at CT are sometimes completely different to the conformers observed at RT. In total, they find that cryo-cooling affects a significant number of residues (predominantly those on the surface of the protein, but also those that are buried). 18.9% of residues have rotamer distributions that change between RT and CT, and 37.7% of residues have a conformer that changes occupancy by 20% or more.
Overall, the authors conclude that, where possible, datasets should be collected at RT, as the derived models offer a more realistic description of the biologically-relevant conformational ensemble of the protein.
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