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- PDB-7ev9: cryoEM structure of particulate methane monooxygenase associated ... -

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Basic information

Entry
Database: PDB / ID: 7ev9
TitlecryoEM structure of particulate methane monooxygenase associated with Cu(I)
Components
  • Ammonia monooxygenase/methane monooxygenase, subunit C family protein
  • Particulate methane monooxygenase alpha subunit
  • Particulate methane monooxygenase beta subunit
KeywordsOXIDOREDUCTASE / particulate methane monooxygenase / copper contained
Function / homology
Function and homology information


methane monooxygenase (particulate) / methane monooxygenase complex / methane monooxygenase activity / methane monooxygenase (soluble) / methane monooxygenase NADH activity / methane monooxygenase NADPH activity / methane metabolic process / monooxygenase activity / membrane / metal ion binding
Similarity search - Function
Ammonia monooxygenase/particulate methane monooxygenase, subunit A / Ammonia monooxygenase/particulate methane monooxygenase, subunit C / Ammonia/methane monooxygenase, subunit B, hairpin domain superfamily / Ammonia/methane monooxygenase, subunit B, C-terminal / Ammonia monooxygenase/particulate methane monooxygenase, subunit C domain superfamily / Ammonia/particulate methane monooxygenase, subunit A superfamily / Ammonia monooxygenase / Ammonia monooxygenase/methane monooxygenase, subunit C / Ammonia monooxygenase/particulate methane monooxygenase, subunit B / Ammonia/methane monooxygenase, subunitB, N-terminal / Monooxygenase subunit B protein
Similarity search - Domain/homology
COPPER (I) ION / Particulate methane monooxygenase alpha subunit / Ammonia monooxygenase/methane monooxygenase, subunit C family protein / Particulate methane monooxygenase beta subunit
Similarity search - Component
Biological speciesMethylococcus capsulatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsChang, W.H. / Lin, H.H. / Tsai, I.K. / Huang, S.H. / Chung, S.C. / Tu, I.P. / Yu, S.F. / Chan, S.I.
CitationJournal: J Am Chem Soc / Year: 2021
Title: Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation.
Authors: W-H Chang / H-H Lin / I-K Tsai / S-H Huang / S-C Chung / I-P Tu / S S-F Yu / S I Chan /
Abstract: The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room ...The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membrane-bound protein with three subunits (PmoB, PmoA, and PmoC) and 12-14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of -pMMO from (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear Cu at the site, resolve two Cus at the site, and uncover additional Cu clusters at the PmoA/PmoC interface within the membrane ( site) and in the water-exposed -terminal subdomain of the PmoB ( clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.
History
DepositionMay 20, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 21, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Particulate methane monooxygenase alpha subunit
B: Particulate methane monooxygenase beta subunit
C: Ammonia monooxygenase/methane monooxygenase, subunit C family protein
E: Particulate methane monooxygenase alpha subunit
F: Particulate methane monooxygenase beta subunit
G: Ammonia monooxygenase/methane monooxygenase, subunit C family protein
I: Particulate methane monooxygenase alpha subunit
J: Particulate methane monooxygenase beta subunit
K: Ammonia monooxygenase/methane monooxygenase, subunit C family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)314,76833
Polymers313,2429
Non-polymers1,52524
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, ab-initio Reconstruction
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Particulate methane monooxygenase alpha subunit / Methane monooxygenase B subunit / Particulate methane monooxygenase 45 kDa subunit / Particulate ...Methane monooxygenase B subunit / Particulate methane monooxygenase 45 kDa subunit / Particulate methane monooxygenase 47 kDa subunit / Particulate methane monooxygenase hydroxylase 45 kDa subunit / Particulate methane monooxygenase hydroxylase alpha subunit / pMMO-H alpha subunit


Mass: 46129.746 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Methylococcus capsulatus (strain ATCC 33009 / NCIMB 11132 / Bath) (bacteria)
Strain: ATCC 33009 / NCIMB 11132 / Bath
References: UniProt: G1UBD1, methane monooxygenase (particulate)
#2: Protein Particulate methane monooxygenase beta subunit / Methane monooxygenase A subunit / Particulate methane monooxygenase 27 kDa subunit / Particulate ...Methane monooxygenase A subunit / Particulate methane monooxygenase 27 kDa subunit / Particulate methane monooxygenase hydroxylase 26 kDa subunit / Particulate methane monooxygenase hydroxylase beta subunit / pMMO-H beta subunit


Mass: 28445.098 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Methylococcus capsulatus (strain ATCC 33009 / NCIMB 11132 / Bath) (bacteria)
Strain: ATCC 33009 / NCIMB 11132 / Bath
References: UniProt: Q607G3, methane monooxygenase (particulate)
#3: Protein Ammonia monooxygenase/methane monooxygenase, subunit C family protein / Methane monooxygenase / C subunit


Mass: 29839.309 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Methylococcus capsulatus (strain ATCC 33009 / NCIMB 11132 / Bath) (bacteria)
Strain: ATCC 33009 / NCIMB 11132 / Bath
References: UniProt: Q603F1, methane monooxygenase (soluble)
#4: Chemical...
ChemComp-CU1 / COPPER (I) ION / Copper


Mass: 63.546 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Cu / 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: particulate methane monooxygenase (pMMO) / Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.312 MDa / Experimental value: NO
Source (natural)Organism: Methylococcus capsulatus str. Bath (bacteria) / Cellular location: membrane
Buffer solutionpH: 7.2
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingMaterial: vitrified ice
VitrificationCryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 278 K

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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: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 23484
Image scansMovie frames/image: 80 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategory
4Gctf1.18CTF correction
10cryoSPARC2.15initial Euler assignment
11RELION3final Euler assignment
12cryoSPARC2.15final Euler assignment
14cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128116 / Symmetry type: POINT

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