Beale Emma V, Warren Anna J, Trincão José, Beilsten-Edmands James, Crawshaw Adam D, Sutton Geoff, Stuart David, Evans Gwyndaf
Life Science, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK.
Division of Structural Biology, University of Oxford, Wellcome Centre for Human Genetics, Oxford, Oxfordshire OX3 7BN, UK.
IUCrJ. 2020 Apr 10;7(Pt 3):500-508. doi: 10.1107/S2052252520003875. eCollection 2020 May 1.
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 , 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.
近年来，开发从微米甚至亚微米大小的蛋白质晶体中确定高分辨率结构的方法变得越来越重要。这适用于大型蛋白质复合物和膜蛋白，在这些情况下，蛋白质生产以及随后大型均匀晶体的生长通常具有挑战性，也适用于仅产生微米或纳米晶体的样品，如淀粉样蛋白或病毒多角体蛋白。钻石光源的通用大分子晶体学微聚焦（VMXm）光束线专门用于对微米和纳米晶体进行X射线衍射测量。由于有可能从接近可见光显微镜分辨率极限的晶体样品中测量数据，光束线设计包括一台扫描电子显微镜（SEM），用于可视化、定位晶体并将其精确置于X射线衍射实验的中心位置。为确保扫描电子显微镜是用于样品可视化的合适方法，进行了测试以评估SEM辐射对衍射质量的影响。在收集X射线衍射数据之前，将在电子显微镜网格上冷冻冷却的细胞质多角体病毒多角体蛋白晶体暴露于SEM辐射下。在用 处理数据后，发现从暴露于和未暴露于SEM辐射的晶体收集的数据集中，数据质量没有统计学上的显著差异。这项研究支持将SEM用作蛋白质晶体可视化的工具以及VMXm光束线上的集成可视化工具。
