Approaches to altering particle distributions in cryo-electron microscopy sample preparation

被引:93
|
作者
Drulyte, Ieva [1 ,2 ]
Johnson, Rachel M. [2 ,3 ,4 ]
Hesketh, Emma L. [1 ,2 ]
Hurdiss, Daniel L. [1 ,2 ]
Scarff, Charlotte A. [1 ,2 ]
Porav, Sebastian A. [5 ]
Ranson, Neil A. [1 ,2 ]
Muench, Stephen P. [2 ,3 ]
Thompson, Rebecca F. [1 ,2 ]
机构
[1] Univ Leeds, Fac Biol Sci, Sch Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England
[2] Univ Leeds, Astbury Ctr Struct & Mol Biol, Leeds LS2 9JT, W Yorkshire, England
[3] Univ Leeds, Fac Biol Sci, Sch Biomed Sci, Leeds LS2 9JT, W Yorkshire, England
[4] Univ Leeds, Fac Math & Phys Chem, Sch Chem, Leeds LS2 9JT, W Yorkshire, England
[5] Natl Inst Res & Dev Isotop & Mol Technol, 67-103 Donat, Cluj Napoca 400293, Romania
基金
英国惠康基金; 英国生物技术与生命科学研究理事会;
关键词
cryo-electron microscopy; single-particle sample preparation; single-particle analysis; CRYO-EM STRUCTURES; CARBON-FILMS; MACROMOLECULAR COMPLEXES; ELECTRON CRYOMICROSCOPY; SUBSTRATE; SPECIMEN; REVEALS; ARCHITECTURE; STABILITY; FILAMENTS;
D O I
10.1107/S2059798318006496
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:560 / 571
页数:12
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