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Title: Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination
In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 µm wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM's widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM 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 cryoEM 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 Å resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.  more » « less
Award ID(s):
2046778
PAR ID:
10493385
Author(s) / Creator(s):
; ;
Publisher / Repository:
JoVE
Date Published:
Journal Name:
Journal of Visualized Experiments
Issue:
201
ISSN:
1940-087X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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