Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air–water interface, the presence of preferred orie...

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Bibliographic Details
Published in:ACS nano 2023-03, Vol.17 (6), p.6011-6022
Main Authors: Ahn, Eungjin, Kim, Byungchul, Park, Soyoung, Erwin, Amanda L., Sung, Suk Hyun, Hovden, Robert, Mosalaganti, Shyamal, Cho, Uhn-Soo
Format: Article
Language:English
Subjects:
Chemistry
Cryoelectron Microscopy - methods
Graphite - chemistry
Materials Science
Methylococcus capsulatus
Proteins
Science & Technology - Other Topics
Water
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Summary:Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air–water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air–water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.
ISSN:1936-0851
1936-086X
DOI:10.1021/acsnano.3c00463