低温电子层析成像
低温电子显微
电子断层摄影术
计算机科学
单粒子分析
软件
断层摄影术
生物系统
人工智能
物理
电子显微镜
光学
生物
核磁共振
气溶胶
程序设计语言
扫描透射电子显微镜
气象学
作者
Tanmay A. M. Bharat,Sjors H. W. Scheres
出处
期刊:Nature Protocols
[Springer Nature]
日期:2016-09-29
卷期号:11 (11): 2054-2065
被引量:251
标识
DOI:10.1038/nprot.2016.124
摘要
Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis. We describe herein a protocol for subtomogram averaging from cryo-ET data using the RELION software (http://www2.mrc-lmb.cam.ac.uk/relion). RELION was originally developed for cryo-EM single-particle analysis, and the subtomogram averaging approach presented in this protocol has been implemented in the existing workflow for single-particle analysis so that users may conveniently tap into existing capabilities of the RELION software. We describe how to calculate 3D models for the contrast transfer function (CTF) that describe the transfer of information in the imaging process, and we illustrate the results of classification and subtomogram averaging refinement for cryo-ET data of purified hepatitis B capsid particles and Saccharomyces cerevisiae 80S ribosomes. Using the steps described in this protocol, along with the troubleshooting and optimization guidelines, high-resolution maps can be obtained in which secondary structure elements are resolved subtomogram.
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