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- EMDB-40719: particulate methane monooxygeanse treated with potassium cyanide ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40719
Titleparticulate methane monooxygeanse treated with potassium cyanide and copper reloaded
Map data
Sample
  • Complex: particulate methane monooxygenaseMethane monooxygenase (particulate)
    • Protein or peptide: Particulate methane monooxygenase alpha subunit
    • Protein or peptide: Particulate methane monooxygenase beta subunit
    • Protein or peptide: Ammonia monooxygenase/methane monooxygenase, subunit C family protein
  • Ligand: COPPER (II) ION
KeywordsMetalloenzyme / Membrane Protein / Nanodiscs / Oxidoreductase
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsTucci FJ / Jodts RJ / Rosenzweig AC
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118035 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM111097 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM105538 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)F31ES034283 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008382 United States
National Science Foundation (NSF, United States)MCB-1908587 United States
CitationJournal: Nat Catal / Year: 2023
Title: Product analog binding identifies the copper active site of particulate methane monooxygenase.
Authors: Frank J Tucci / Richard J Jodts / Brian M Hoffman / Amy C Rosenzweig /
Abstract: Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyzes the oxidation of methane to methanol. pMMO activity requires copper, and decades of ...Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyzes the oxidation of methane to methanol. pMMO activity requires copper, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the Cu, Cu, and Cu sites. Challenges associated with the isolation of active pMMO have hindered progress toward locating its catalytic center. However, reconstituting pMMO into native lipid nanodiscs stabilizes its structure and recovers its activity. Here, these active samples were incubated with 2,2,2,-trifluoroethanol (TFE), a product analog that serves as a readily visualized active-site probe. Interactions of TFE with the Cu site were observed by both pulsed ENDOR spectroscopy and cryoEM, implicating Cu and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoEM maps.
History
DepositionMay 5, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40719.png

Downloads & links

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Map

FileDownload / File: emd_40719.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.5291 Å
Density
Contour LevelBy AUTHOR: 0.119
Minimum - Maximum-0.4180472 - 0.78600204
Average (Standard dev.)0.0029230185 (±0.023669364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 270.8992 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40719_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40719_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : particulate methane monooxygenase

EntireName: particulate methane monooxygenaseMethane monooxygenase (particulate)
Components
  • Complex: particulate methane monooxygenaseMethane monooxygenase (particulate)
    • Protein or peptide: Particulate methane monooxygenase alpha subunit
    • Protein or peptide: Particulate methane monooxygenase beta subunit
    • Protein or peptide: Ammonia monooxygenase/methane monooxygenase, subunit C family protein
  • Ligand: COPPER (II) ION

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Supramolecule #1: particulate methane monooxygenase

SupramoleculeName: particulate methane monooxygenase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Methylococcus capsulatus (bacteria) / Strain: Bath
Molecular weightTheoretical: 337.11 kDa/nm

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Macromolecule #1: Particulate methane monooxygenase alpha subunit

MacromoleculeName: Particulate methane monooxygenase alpha subunit / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Methylococcus capsulatus (bacteria) / Strain: Bath
Molecular weightTheoretical: 42.832887 KDa
SequenceString: HGEKSQAAFM RMRTIHWYDL SWSKEKVKIN ETVEIKGKFH VFEGWPETVD EPDVAFLNVG MPGPVFIRKE SYIGGQLVPR SVRLEIGKT YDFRVVLKAR RPGDWHVHTM MNVQGGGPII GPGKWITVEG SMSEFRNPVT TLTGQTVDLE NYNEGNTYFW H AFWFAIGV ...String:
HGEKSQAAFM RMRTIHWYDL SWSKEKVKIN ETVEIKGKFH VFEGWPETVD EPDVAFLNVG MPGPVFIRKE SYIGGQLVPR SVRLEIGKT YDFRVVLKAR RPGDWHVHTM MNVQGGGPII GPGKWITVEG SMSEFRNPVT TLTGQTVDLE NYNEGNTYFW H AFWFAIGV AWIGYWSRRP IFIPRLLMVD AGRADELVSA TDRKVAMGFL AATILIVVMA MSSANSKYPI TIPLQAGTMR GM KPLELPA PTVSVKVEDA TYRVPGRAMR MKLTITNHGN SPIRLGEFYT ASVRFLDSDV YKDTTGYPED LLAEDGLSVS DNS PLAPGE TRTVDVTASD AAWEVYRLSD IIYDPDSRFA GLLFFFDATG NRQVVQIDAP LIPSFM

UniProtKB: Particulate methane monooxygenase alpha subunit

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Macromolecule #2: Particulate methane monooxygenase beta subunit

MacromoleculeName: Particulate methane monooxygenase beta subunit / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO / EC number: methane monooxygenase (particulate)
Source (natural)Organism: Methylococcus capsulatus (bacteria) / Strain: Bath
Molecular weightTheoretical: 28.445098 KDa
SequenceString: MSAAQSAVRS HAEAVQVSRT IDWMALFVVF FVIVGSYHIH AMLTMGDWDF WSDWKDRRLW VTVTPIVLVT FPAAVQSYLW ERYRLPWGA TVCVLGLLLG EWINRYFNFW GWTYFPINFV FPASLVPGAI ILDTVLMLSG SYLFTAIVGA MGWGLIFYPG N WPIIAPLH ...String:
MSAAQSAVRS HAEAVQVSRT IDWMALFVVF FVIVGSYHIH AMLTMGDWDF WSDWKDRRLW VTVTPIVLVT FPAAVQSYLW ERYRLPWGA TVCVLGLLLG EWINRYFNFW GWTYFPINFV FPASLVPGAI ILDTVLMLSG SYLFTAIVGA MGWGLIFYPG N WPIIAPLH VPVEYNGMLM SIADIQGYNY VRTGTPEYIR MVEKGTLRTF GKDVAPVSAF FSAFMSILIY FMWHFIGRWF SN ERFLQST

UniProtKB: Particulate methane monooxygenase beta subunit

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Macromolecule #3: Ammonia monooxygenase/methane monooxygenase, subunit C family protein

MacromoleculeName: Ammonia monooxygenase/methane monooxygenase, subunit C family protein
type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Methylococcus capsulatus (bacteria) / Strain: Bath
Molecular weightTheoretical: 27.65684 KDa
SequenceString: LLDKKWLTFA LAIYTVFYLW VRWYEGVYGW SAGLDSFAPE FETYWMNFLY TEIVLEIVTA SILWGYLWKT RDRNLAALTP REELRRNFT HLVWLVAYAW AIYWGASYFT EQDGTWHQTI VRDTDFTPSH IIEFYLSYPI YIITGFAAFI YAKTRLPFFA K GISLPYLV ...String:
LLDKKWLTFA LAIYTVFYLW VRWYEGVYGW SAGLDSFAPE FETYWMNFLY TEIVLEIVTA SILWGYLWKT RDRNLAALTP REELRRNFT HLVWLVAYAW AIYWGASYFT EQDGTWHQTI VRDTDFTPSH IIEFYLSYPI YIITGFAAFI YAKTRLPFFA K GISLPYLV LVVGPFMILP NVGLNEWGHT FWFMEELFVA PLHYGFVIFG WLALAVMGTL TQTFYSFAQG GLGQSLCE

UniProtKB: Ammonia monooxygenase/methane monooxygenase, subunit C family protein

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Macromolecule #4: COPPER (II) ION

MacromoleculeName: COPPER (II) ION / type: ligand / ID: 4 / Number of copies: 9 / Formula: CU
Molecular weightTheoretical: 63.546 Da
Chemical component information

ChemComp-CU:
COPPER (II) ION / Copper

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.3
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.4 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.56 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7s4h

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 864874

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