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使用 RELION 中的子断层平均法从电子冷冻断层扫描数据中解析大分子结构。

Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION.

机构信息

Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK.

出版信息

Nat Protoc. 2016 Nov;11(11):2054-65. doi: 10.1038/nprot.2016.124. Epub 2016 Sep 29.

DOI:10.1038/nprot.2016.124
PMID:27685097
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5215819/
Abstract

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.

摘要

电子低温断层扫描（cryo-ET）是一种技术，用于从一系列倾斜的电子低温显微镜（cryo-EM）图像中生成复杂物体（如不对称病毒、细胞细胞器或整个细胞）的 3D 图片（断层扫描）。在断层扫描中发现的大分子复合物的平均称为亚断层扫描平均，该技术允许原位确定大分子复合物的结构。亚断层扫描平均也越来越流行，用于计算单颗粒分析的初始模型。我们在此描述了一种使用 RELION 软件（http://www2.mrc-lmb.cam.ac.uk/relion）从 cryo-ET 数据进行亚断层扫描平均的方案。RELION 最初是为 cryo-EM 单颗粒分析开发的，本方案中提出的亚断层扫描平均方法已在现有的单颗粒分析工作流程中实现，以便用户可以方便地利用 RELION 软件的现有功能。我们描述了如何计算描述成像过程中信息传递的对比度传递函数（CTF）的 3D 模型，并展示了纯化乙型肝炎外壳颗粒和酿酒酵母 80S 核糖体的 cryo-ET 数据的分类和亚断层扫描平均细化的结果。使用本方案中描述的步骤以及故障排除和优化指南，可以获得高分辨率图谱，其中亚断层扫描可以分辨二级结构元素。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5b9c/5215819/50bc6c196dc6/emss-70850-f006.jpg
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