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bioRxiv · 10.1101/2023.07.28.550908

Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination

Grassetti, A. V.·May, M. B.·Davis, J. H.
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Abstract

In cryogenic electron microscopy (cryo-EM), purified macromolecules are typically applied to a grid bearing a holey carbon foil, blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice that is suspended across roughly 1 m-wide foil holes. The resulting sample is then imaged using cryogenic transmission electron microscopy and, after substantial image processing, near-atomic resolution structures can be determined. Despite cryo-EMs widespread adoption, sample preparation remains a severe bottleneck in cryo-EM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryo-EM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryo-EM grids, and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 [A] resolution density map of an exemplar Cas9 complex using a highly pure sample at a relatively low concentration. SUMMARYThe application of support layers, such as graphene, to cryo-electron microscopy grids can increase the density of particles imaged, limit particle interactions with the air-water interface, reduce the extent of beam-induced motion, and, in some instances, improve the distribution of particle orientations. This paper describes a robust protocol for coating cryo-EM grids with a monolayer of graphene for improved cryo-sample preparation.

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Grassetti, A. V., May, M. B., Davis, J. H.. 2023-07-28. Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination. https://doi.org/10.1101/2023.07.28.550908

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