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- PDB-7tc7: Cryo-EM structure of methane monooxygenase hydroxylase (by quantifoil) -

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Basic information

Entry
Database: PDB / ID: 7tc7
TitleCryo-EM structure of methane monooxygenase hydroxylase (by quantifoil)
Components
  • Methane monooxygenase component A alpha chain
  • Methane monooxygenase component A beta chain
  • Methane monooxygenase component A gamma chain
KeywordsOXIDOREDUCTASE / methane monooxygenase / hydroxylase / cryo-EM / electron transport
Function / homology
Function and homology information


methane monooxygenase (soluble) / methane monooxygenase NADH activity / methane monooxygenase NADPH activity / methane metabolic process / : / one-carbon metabolic process / metal ion binding
Similarity search - Function
Methane monooxygenase, gamma chain / Methane monooxygenase, gamma chain, domain 1 / Methane monooxygenase, gamma chain, domain 2 / Methane monooxygenase, gamma chain superfamily / Methane monooxygenase, hydrolase gamma chain / Methane/phenol monooxygenase, hydroxylase component / Propane/methane/phenol/toluene hydroxylase / Methane/Phenol/Alkene Hydroxylase / Ribonucleotide reductase-like / Ferritin-like superfamily
Similarity search - Domain/homology
: / Methane monooxygenase component A gamma chain / Methane monooxygenase component A beta chain / Methane monooxygenase component A alpha chain
Similarity search - Component
Biological speciesMethylococcus capsulatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsCho, U.S. / Kim, B.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK111465 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA250329 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS116008 United States
CitationJournal: ACS Nano / Year: 2023
Title: Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Soluble Methane Monooxygenase Hydroxylase.
Authors: Eungjin Ahn / Byungchul Kim / Soyoung Park / Amanda L Erwin / Suk Hyun Sung / Robert Hovden / Shyamal Mosalaganti / Uhn-Soo Cho /
Abstract: 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 ...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.
History
DepositionDec 23, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Apr 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3May 17, 2023Group: Database references / Category: citation_author / Item: _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: Methane monooxygenase component A alpha chain
E: Methane monooxygenase component A alpha chain
B: Methane monooxygenase component A beta chain
C: Methane monooxygenase component A beta chain
G: Methane monooxygenase component A gamma chain
H: Methane monooxygenase component A gamma chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,78610
Polymers251,5636
Non-polymers2234
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Methane monooxygenase component A alpha chain / / Methane hydroxylase


Mass: 60717.145 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Methylococcus capsulatus (bacteria) / Strain: ATCC 33009 / NCIMB 11132 / Bath
References: UniProt: P22869, methane monooxygenase (soluble)
#2: Protein Methane monooxygenase component A beta chain / / Methane hydroxylase


Mass: 45184.660 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Methylococcus capsulatus (bacteria)
References: UniProt: P18798, methane monooxygenase (soluble)
#3: Protein Methane monooxygenase component A gamma chain / / Methane hydroxylase


Mass: 19879.732 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Methylococcus capsulatus (bacteria) / Strain: ATCC 33009 / NCIMB 11132 / Bath
References: UniProt: P11987, methane monooxygenase (soluble)
#4: Chemical
ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: soluble methane monooxygenase hydroxylase / Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Methylococcus capsulatus str. Bath (bacteria)
Source (recombinant)Organism: Methylococcus capsulatus str. Bath (bacteria)
Buffer solutionpH: 7 / Details: 30 mM HEPES, pH 7, 150 mM NaCl, 1 mM TCEP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 479000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00517865
ELECTRON MICROSCOPYf_angle_d0.50124241
ELECTRON MICROSCOPYf_dihedral_angle_d4.8222343
ELECTRON MICROSCOPYf_chiral_restr0.0412463
ELECTRON MICROSCOPYf_plane_restr0.0043155

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