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bioRxiv · 10.1101/827089

High Yield Monolayer Graphene Grids for Near-Atomic Resolution Cryo-Electron Microscopy

Han, Y.·Fan, X.·Wang, H.·Zhao, F.·Tully, C. G.·Kong, J.·Yao, N.·Yan, N.
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Abstract

Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids--aimed at preserving thin, uniform vitrified ice and improving protein adsorption--have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6 [A] resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipo-proteins by at least five times, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, our approach allows for customized grid designs targeting specific proteins, due to its broad compatibility with a variety of nanomaterials. Significance statementSingle particle cryogenic electron microscopy (cryo-EM) represents the cutting-edge technology to determine three-dimensional atomic structures of bio-macromolecules. However, issues of cryo-sample preparation limit the cryo-EM to achieve higher resolution. Here, we demonstrated a high yield, monolayer graphene supporting film to improve the cryo-sample quality. Using our approach, we have achieved so far, the highest resolution structure of the smallest protein by cryo-EM with the minimal number of datasets. Our technique paves the way for universal cryo-sample preparation for near-atomic resolution cryo-EM.

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Han, Y., Fan, X., Wang, H., Zhao, F., Tully, C. G., Kong, J., Yao, N., Yan, N.. 2019-11-01. High Yield Monolayer Graphene Grids for Near-Atomic Resolution Cryo-Electron Microscopy. https://doi.org/10.1101/827089

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