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bioRxiv · 10.1101/2020.03.17.995852

A Robust Method for Collecting X-ray Diffraction Data from Protein Crystals across Physiological Temperatures

Doukov, T.·Herschlag, D.·Yabukarski, F.
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Abstract

Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure-function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Further, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that is directly related to biological function. The next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperatures and was addressed herein. This approach provides complete diffraction datasets with total collection times as short as ~5 sec from single protein crystals, dramatically increasing the amount of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 [A] resolution data over a temperature range of 293-363 K for proteinase K, thaumatin, and lysozyme crystals. Our analyses indicate that the diffraction data is of high-quality and do not suffer from excessive dehydration or damage.

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Doukov, T., Herschlag, D., Yabukarski, F.. 2020-03-17. A Robust Method for Collecting X-ray Diffraction Data from Protein Crystals across Physiological Temperatures. https://doi.org/10.1101/2020.03.17.995852

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