High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

被引：95

作者：

Moriya, Toshio ^{[1
]}

Saur, Michael ^{[1
]}

Stabrin, Markus ^{[1
]}

Merino, Felipe ^{[1
]}

Voicu, Horatiu ^{[2
]}

Huang, Zhong ^{[2
]}

Penczek, Pawel A. ^{[2
]}

Raunser, Stefan ^{[1
]}

Gatsogiannis, Christos ^{[1
]}

机构：

[1] Max Planck Inst Mol Physiol, Dept Struct Biochem, Dortmund, Germany

[2] Univ Texas Med Sch Houston, Dept Biochem & Mol Biol, Houston, TX USA

来源：

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2017年 / 123期

基金：

美国国家卫生研究院;

关键词：

Biochemistry; Issue; 123; structural biology; electron microscopy; electron cryo-microscopy; cryo-EM; TEM; single particle image processing; software development; single particle analysis; Tc toxin; CRYO-EM STRUCTURE; MECHANISM; COMPLEX;

D O I：

10.3791/55448

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 angstrom structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

引用

页数：11