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TitleBatch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Soluble Methane Monooxygenase Hydroxylase.
Journal, issue, pagesACS Nano, Vol. 17, Issue 6, Page 6011-6022, Year 2023
Publish dateMar 28, 2023
AuthorsEungjin Ahn / Byungchul Kim / Soyoung Park / Amanda L Erwin / Suk Hyun Sung / Robert Hovden / Shyamal Mosalaganti / Uhn-Soo Cho /
PubMed AbstractCryogenic 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 ...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 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.
External linksACS Nano / PubMed:36926824 / PubMed Central
MethodsEM (single particle)
Resolution2.4 - 2.9 Å
Structure data

25804

7tc7

EMDB-25804, PDB-7tc7:
Cryo-EM structure of methane monooxygenase hydroxylase (by quantifoil)
Method: EM (single particle) / Resolution: 2.9 Å

25805

7tc8

EMDB-25805, PDB-7tc8:
Cryo-EM structure of methane monooxygenase hydroxylase (by graphene)
Method: EM (single particle) / Resolution: 2.4 Å

Chemicals

ChemComp-FE:
Unknown entry / Iron

Source
  • Methylococcus capsulatus str. Bath (bacteria)
  • methylococcus capsulatus (bacteria)
KeywordsOXIDOREDUCTASE / methane monooxygenase / hydroxylase / cryo-EM / electron transport

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