Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures

Tzanko Doukov; Daniel Herschlag; Filip Yabukarski

doi:10.1107/S1600576720013503

Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures

J Appl Crystallogr. 2020 Nov 5;53(Pt 6):1493-1501. doi: 10.1107/S1600576720013503. eCollection 2020 Dec 1.

Authors

Tzanko Doukov¹, Daniel Herschlag^{2

3

4}, Filip Yabukarski²

Affiliations

¹ SMB, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
² Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
³ Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA.
⁴ Stanford ChEM-H, Stanford University, Stanford, CA 94305, USA.

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. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as ∼5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 Å resolution data over a temperature range of 293-363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.

Keywords: X-ray crystallography; data collection methods; physiological temperature; protein crystals; room temperature.

Grants and funding

P41 GM103393/GM/NIGMS NIH HHS/United States