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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Published inProceedings of the National Academy of Sciences - PNAS Vol. 117; no. 39; pp. 24269 - 24273
Main Authors Wang, Feng, Liu, Yanxin, Yu, Zanlin, Li, Sam, Feng, Shengjie, Cheng, Yifan, Agard, David A.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 29.09.2020
Subjects
Affinity
Air-water interface
Biological Sciences
Covalent bonds
Cryoelectron Microscopy - methods
Denaturation
Electron microscopy
Graphene
Graphite
High resolution
Polyethylene glycol
Polyethylene Glycols
Specimen Handling - methods
affinity grid
graphene oxide
cryo-EM
single-particle reconstruction
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Summary: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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Contributed by David A. Agard, July 2, 2020 (sent for review May 16, 2020; reviewed by John L. Rubinstein and Hongwei Wang)
Author contributions: F.W., Y.C., and D.A.A. designed research; F.W., Y.L., Z.Y., S.L., and S.F. performed research; F.W., Y.L., Z.Y., S.L., and S.F. analyzed data; F.W., Y.L., Z.Y., S.L., and S.F. wrote the paper with input from Y.C.; and Y.C. and D.A.A. supervised the project.
Reviewers: J.L.R., The Hospital for Sick Children Research Institute; and H.W., Tsinghua University.
1Y.L. and Z.Y. contributed equally to this work.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.2009707117