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

Emma V Beale,James Beilsten-Edmands,José Trincão,Adam D Crawshaw,David Stuart,Anna J Warren,Geoff Sutton,Gwyndaf Evans

doi:10.1107/s2052252520003875

Emma V Beale, James Beilsten-Edmands + Show 6 more

Open Access

https://doi.org/10.1107/s2052252520003875

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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.

Highlights

In the last decade, microfocus X-ray beamlines have facilitated advances in structural biology by providing increasingly small intense beams of X-rays
The analyses described here support the use of low-voltage scanning electron microscope (SEM) imaging as a method to visualize and locate micrometresized protein crystals prior to X-ray diffraction experiments
Using 2 keV electrons at the doses described, the results presented here indicate no significant difference between the quality of X-ray diffraction data from crystals that were exposed to the SEM beam and those that were not

Summary

Introduction

Microfocus X-ray beamlines have facilitated advances in structural biology by providing increasingly small intense beams of X-rays. Crystal sizes on the order of tens of micrometres down to a few micrometres are generally considered accessible, albeit challenging, targets for protein structural biology projects. Serial femtosecond crystallography X-ray free-electron laser (XFEL) approaches have pushed this limit, using tens of thousands of microcrystals [for a review see Martin-Garcia et al (2016)] and even nanocrystals (Gati et al, 2017) to determine high-resolution protein structures. Synchrotron serial crystallography methods are developing, but again often require reasonably large numbers of crystals (Ebrahim et al, 2019; Diederichs & Wang, 2017). Electron diffraction is another growing technique for structure determination from protein crystals that are a few hundred

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