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- PDB-9pxf: Ammonia monooxygenase in native membranes from N. briensis -

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Basic information

Entry
Database: PDB / ID: 9pxf
TitleAmmonia monooxygenase in native membranes from N. briensis
Components(Ammonia monooxygenase subunit ...) x 3
KeywordsOXIDOREDUCTASE / ammonia oxidation / copper enzyme / membrane protein / active site
Function / homology
Function and homology information


monooxygenase activity / membrane
Similarity search - Function
Ammonia monooxygenase/particulate methane monooxygenase, subunit A / Ammonia/methane monooxygenase, subunit B, hairpin domain superfamily / Ammonia/methane monooxygenase, subunit B, C-terminal / Ammonia/particulate methane monooxygenase, subunit A superfamily / Ammonia monooxygenase / Ammonia monooxygenase/particulate methane monooxygenase, subunit B / Ammonia/methane monooxygenase, subunitB, N-terminal / Monooxygenase subunit B protein
Similarity search - Domain/homology
Chem-6PL / COPPER (II) ION / : / Ammonia monooxygenase subunit A / Ammonia monooxygenase subunit B
Similarity search - Component
Biological speciesNitrosospira briensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.58 Å
AuthorsFrank, F.J. / Rosenzweig, A.C.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118035 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)T32GM105538 United States
Department of Energy (DOE, United States)DE-SC0016284 United States
CitationJournal: Chem Sci / Year: 2025
Title: Simultaneous occupancy of Cu and Cu in the ammonia monooxygenase active site.
Authors: Frank J Tucci / Madeline B Ho / Aaron A B Turner / Lisa Y Stein / Brian M Hoffman / Amy C Rosenzweig /
Abstract: Ammonia monooxygenase (AMO), a copper-dependent membrane enzyme, catalyzes the first and rate-limiting step of nitrification: the oxidation of ammonia to hydroxylamine. Despite its central role in ...Ammonia monooxygenase (AMO), a copper-dependent membrane enzyme, catalyzes the first and rate-limiting step of nitrification: the oxidation of ammonia to hydroxylamine. Despite its central role in the global nitrogen cycle and its biotechnological relevance, structural characterization of AMO has lagged behind that of its homolog, particulate methane monooxygenase (pMMO), due to the slow growth rates of ammonia-oxidizing bacteria and the instability of AMO upon purification. Recent cryoEM studies of AMO and (Bath) pMMO in native membranes revealed new structural features, including two adjacent copper-binding sites in the transmembrane region, Cu and Cu, believed to constitute the active site. Although multiple structures were determined under various conditions, simultaneous occupancy of Cu and Cu was never observed, leaving their potential functional interplay unresolved. Here we report the 2.6 Å resolution cryoEM structure of AMO from C-128 in isolated native membranes. This structure reveals the first instance of simultaneous copper occupancy of the Cu and Cu sites, along with occupancy of the periplasmic Cu site. Electron paramagnetic resonance (EPR) spectroscopic data indicate that the Cu site is primarily occupied by Cu(ii), while Cu and Cu are primarily occupied by diamagnetic ions, presumably Cu(i). Notably, a lipid molecule is bound between the Cu and Cu sites, separating them by ∼8.0 Å. The results underscore the importance of studying these enzymes in their native environments across species to resolve conserved and divergent molecular features.
History
DepositionAug 5, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 28, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Ammonia monooxygenase subunit C
D: Ammonia monooxygenase subunit A
A: Ammonia monooxygenase subunit B
E: Ammonia monooxygenase subunit A
F: Ammonia monooxygenase subunit A
B: Ammonia monooxygenase subunit B
C: Ammonia monooxygenase subunit B
H: Ammonia monooxygenase subunit C
I: Ammonia monooxygenase subunit C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)313,35621
Polymers310,4949
Non-polymers2,86112
Water15,277848
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Ammonia monooxygenase subunit ... , 3 types, 9 molecules GHIDEFABC

#1: Protein Ammonia monooxygenase subunit C


Mass: 29534.312 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrosospira briensis (bacteria) / Gene: SAMN05720354_1358 / Production host: Nitrosospira briensis (bacteria) / References: UniProt: A0A1G4ZCX6
#2: Protein Ammonia monooxygenase subunit A


Mass: 31288.760 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrosospira briensis (bacteria) / Gene: SAMN05216386_3025 / Production host: Nitrosospira briensis (bacteria) / References: UniProt: A0A1I5FI55
#3: Protein Ammonia monooxygenase subunit B


Mass: 42675.074 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrosospira briensis (bacteria) / Gene: SAMN05216386_3024 / Production host: Nitrosospira briensis (bacteria) / References: UniProt: A0A1I5FIF1

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Non-polymers , 3 types, 860 molecules

#4: Chemical
ChemComp-CU / COPPER (II) ION


Mass: 63.546 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-6PL / (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / 1-PALMITOYL-2-STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE


Mass: 763.100 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H85NO8P / Comment: phospholipid*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 848 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ammonia monooxygenase in native membranes from N. briensis
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#3 / Source: NATURAL
Source (natural)Organism: Nitrosospira briensis (bacteria)
Buffer solutionpH: 7.2
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212075 / Symmetry type: POINT

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