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Computational separation of conformational heterogeneity using cryo-electron tomography and 3D sub-volume averaging.

Abstract：

:We have previously used cryo-electron tomography combined with sub-volume averaging and classification to obtain 3D structures of macromolecular assemblies in cases where a single dominant species was present, and applied these methods to the analysis of a variety of trimeric HIV-1 and SIV envelope glycoproteins (Env). Here, we extend these studies by demonstrating automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. We present a method for measuring the spatial distribution of the vector elements representing distinct conformational states of Env. We identify data processing strategies that allow clear separation of the previously characterized closed and open conformations, as well as unliganded and antibody-liganded states of Env when they are present in mixtures. We show that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual Env sub-volumes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity in heterogeneous mixtures. We validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded as well as open and closed conformations of Env present simultaneously in mixtures.

journal_name

J Struct Biol

journal_title

Journal of structural biology

authors

Frank GA,Bartesaghi A,Kuybeda O,Borgnia MJ,White TA,Sapiro G,Subramaniam S

doi

10.1016/j.jsb.2012.01.004

subject

Has Abstract

pub_date

2012-05-01 00:00:00

pages

165-76

issue

2

eissn

1047-8477

issn

1095-8657

pii

S1047-8477(12)00006-8

journal_volume

178

pub_type

杂志文章

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