Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 39, Pages 24269-24273Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2009707117
Keywords
cryo-EM; graphene oxide; affinity grid; single-particle reconstruction
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Funding
- University of California San Francisco Program for Breakthrough Biomedical Research technology development grant
- NIH [R01 GM098672, P50 GM082250, P01 GM11126, R35 GM118099, U54 CA209891, U01 MH115747, U19 AI135990, S10 OD020054, S10 OD021741]
- Howard Hughes Medical Institute
- Howard Hughes Medical Institute-Helen Hay Whitney Foundation Postdoctoral Fellowship
- American Heart Association Postdoctoral Fellowship [18POST33990362]
- Program for Breakthrough Biomedical Research - Sandler Foundation
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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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