School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, England.
School of Biomedical Sciences, Faculty of Biological Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, England.
Acta Crystallogr D Struct Biol. 2018 Jun 1;74(Pt 6):560-571. doi: 10.1107/S2059798318006496. Epub 2018 May 18.
Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.
现在可以使用冷冻电子显微镜（cryo-EM）来确定各种生物样本的高分辨率结构信息。最近的进展主要是由于显微镜和探测器的发展，以及图像处理软件的进步。然而，对于许多单颗粒 cryo-EM 项目来说，目前主要的瓶颈仍然存在于样品制备阶段；获得足够高质量的 cryo-EM 网格以进行高分辨率单颗粒分析可能需要仔细优化许多变量。常见的障碍包括与样品本身（缓冲成分、不稳定的复合物）、样品分布（获得正确的浓度、对支撑膜的亲和力）、优选方向以及批次内和批次之间的网格制作过程的可重复性有关的问题。这篇综述概述了电子显微镜界内用于解决这些挑战的一些方法，提供了一系列可能有助于获得最佳网格以进行高分辨率数据收集的方法。
