National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM118099
米国
National Institutes of Health/National Cancer Institute (NIH/NCI)
U54CA209891
米国
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)
U01MH115747
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
U19AI135990
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2020 タイトル: General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM. 著者: Feng Wang / Yanxin Liu / Zanlin Yu / Sam Li / Shengjie Feng / Yifan Cheng / David A Agard / 要旨: 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 ...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.