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General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

Affinity grids have great potential to facilitate rapid preparation of even quite impure samples in single-particle cryo-electron microscopy (EM). Yet despite the promising advances of affinity grids over the past decades, no single strategy has demonstrated general utility. Here we chemically funct...

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Detalles Bibliográficos
Autores principales: Wang, Feng, Liu, Yanxin, Yu, Zanlin, Li, Sam, Feng, Shengjie, Cheng, Yifan, Agard, David A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533693/
https://www.ncbi.nlm.nih.gov/pubmed/32913054
http://dx.doi.org/10.1073/pnas.2009707117
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author Wang, Feng
Liu, Yanxin
Yu, Zanlin
Li, Sam
Feng, Shengjie
Cheng, Yifan
Agard, David A.
author_facet Wang, Feng
Liu, Yanxin
Yu, Zanlin
Li, Sam
Feng, Shengjie
Cheng, Yifan
Agard, David A.
author_sort Wang, Feng
collection PubMed
description Affinity grids have great potential to facilitate rapid preparation of even quite impure samples in single-particle cryo-electron microscopy (EM). Yet despite the promising advances of affinity grids over the past decades, no single strategy has demonstrated general utility. Here we chemically functionalize cryo-EM grids coated with mostly one or two layers of graphene oxide to facilitate affinity capture. The protein of interest is tagged using a system that rapidly forms a highly specific covalent bond to its cognate catcher linked to the grid via a polyethylene glycol (PEG) spacer. Importantly, the spacer keeps particles away from both the air–water interface and the graphene oxide surface, protecting them from potential denaturation and rendering them sufficiently flexible to avoid preferential sample orientation concerns. Furthermore, the PEG spacer successfully reduces nonspecific binding, enabling high-resolution reconstructions from a much cruder lysate sample.
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spelling pubmed-75336932020-10-13 General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM Wang, Feng Liu, Yanxin Yu, Zanlin Li, Sam Feng, Shengjie Cheng, Yifan Agard, David A. Proc Natl Acad Sci U S A Biological Sciences Affinity grids have great potential to facilitate rapid preparation of even quite impure samples in single-particle cryo-electron microscopy (EM). Yet despite the promising advances of affinity grids over the past decades, no single strategy has demonstrated general utility. Here we chemically functionalize cryo-EM grids coated with mostly one or two layers of graphene oxide to facilitate affinity capture. The protein of interest is tagged using a system that rapidly forms a highly specific covalent bond to its cognate catcher linked to the grid via a polyethylene glycol (PEG) spacer. Importantly, the spacer keeps particles away from both the air–water interface and the graphene oxide surface, protecting them from potential denaturation and rendering them sufficiently flexible to avoid preferential sample orientation concerns. Furthermore, the PEG spacer successfully reduces nonspecific binding, enabling high-resolution reconstructions from a much cruder lysate sample. National Academy of Sciences 2020-09-29 2020-09-10 /pmc/articles/PMC7533693/ /pubmed/32913054 http://dx.doi.org/10.1073/pnas.2009707117 Text en Copyright © 2020 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/ https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Wang, Feng
Liu, Yanxin
Yu, Zanlin
Li, Sam
Feng, Shengjie
Cheng, Yifan
Agard, David A.
General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM
title General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM
title_full General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM
title_fullStr General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM
title_full_unstemmed General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM
title_short General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM
title_sort general and robust covalently linked graphene oxide affinity grids for high-resolution cryo-em
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533693/
https://www.ncbi.nlm.nih.gov/pubmed/32913054
http://dx.doi.org/10.1073/pnas.2009707117
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