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Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography

DOI: 10.1107/S2052252520003875 DOI Help

Authors: Emma V. Beale (Diamond Light Source) , Anna J. Warren (Diamond Light Source) , Jose Trincao (Diamond Light Source) , James Beilsten-edmands (Diamond Light Source) , Adam D. Crawshaw (Diamond Light Source) , Geoff Sutton (University of Oxford) , David Stuart (Diamond Light Source; University of Oxford) , Gwyndaf Evans (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 7

State: Published (Approved)
Published: May 2020

Open Access Open Access

Abstract: Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.

Journal Keywords: microfocus X-ray diffraction; VMXm beamline; macromolecular crystallography; cryoEM; structural biology; radiation damage; scanning electron microscopy; visualization tools

Subject Areas: Technique Development, Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Documents:
jt5045.pdf