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Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Abstract：

:Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

journal_name

J Struct Biol

journal_title

Journal of structural biology

authors

Yu G,Vago F,Zhang D,Snyder JE,Yan R,Zhang C,Benjamin C,Jiang X,Kuhn RJ,Serwer P,Thompson DH,Jiang W

doi

10.1016/j.jsb.2014.04.006

subject

Has Abstract

pub_date

2014-07-01 00:00:00

pages

1-9

issue

eissn

1047-8477

issn

1095-8657

pii

S1047-8477(14)00092-6

journal_volume

187

pub_type

杂志文章

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Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.