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- EMDB-71966: Ammonia monooxygenase in native membranes from N. briensis -

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Basic information

Entry
Database: EMDB / ID: EMD-71966
TitleAmmonia monooxygenase in native membranes from N. briensis
Map data
Sample
  • Organelle or cellular component: Ammonia monooxygenase in native membranes from N. briensis
    • Protein or peptide: Ammonia monooxygenase subunit C
    • Protein or peptide: Ammonia monooxygenase subunit A
    • Protein or peptide: Ammonia monooxygenase subunit B
  • Ligand: COPPER (II) ION
  • Ligand: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
  • Ligand: water
Keywordsammonia oxidation / copper enzyme / membrane protein / active site / OXIDOREDUCTASE
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
: / Ammonia monooxygenase subunit A / Ammonia monooxygenase subunit B
Similarity search - Component
Biological speciesNitrosospira briensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.58 Å
AuthorsFrank FJ / Rosenzweig AC
Funding support United States, 4 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 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, 2025-
Header (metadata) releaseJan 28, 2026-
Map releaseJan 28, 2026-
UpdateJan 28, 2026-
Current statusJan 28, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71966.png

Downloads & links

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Map

FileDownload / File: emd_71966.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.42 Å/pix.
x 640 pix.
= 270.208 Å		0.42 Å/pix.
x 640 pix.
= 270.208 Å		0.42 Å/pix.
x 640 pix.
= 270.208 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.4222 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0191
Minimum - Maximum-0.13302457 - 0.17802149
Average (Standard dev.)0.00021373248 (±0.0047728396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 270.208 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_71966_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_71966_half_map_2.map
Projections & Slices
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Sample components

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Entire : Ammonia monooxygenase in native membranes from N. briensis

EntireName: Ammonia monooxygenase in native membranes from N. briensis
Components
  • Organelle or cellular component: Ammonia monooxygenase in native membranes from N. briensis
    • Protein or peptide: Ammonia monooxygenase subunit C
    • Protein or peptide: Ammonia monooxygenase subunit A
    • Protein or peptide: Ammonia monooxygenase subunit B
  • Ligand: COPPER (II) ION
  • Ligand: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
  • Ligand: water

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Supramolecule #1: Ammonia monooxygenase in native membranes from N. briensis

SupramoleculeName: Ammonia monooxygenase in native membranes from N. briensis
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Nitrosospira briensis (bacteria)

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Macromolecule #1: Ammonia monooxygenase subunit C

MacromoleculeName: Ammonia monooxygenase subunit C / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Nitrosospira briensis (bacteria)
Molecular weightTheoretical: 29.534312 KDa
Recombinant expressionOrganism: Nitrosospira briensis (bacteria)
SequenceString: DMSEWYDSRW YKFGLITMLL VAIFWVWYQR TYAYSHGMDS MEPEFEKVWM GLWRVHMIVM PIFALITWGW IWKTRDTNLD NLDPKLEVK RYFYWMMWLG VYLFGVYWGG SFFTEQDASW HQVIIRDTSF TPSHVVVFYG SFPMYIVCGV ASYLYAMTRL P LYSRGTSF ...String:
DMSEWYDSRW YKFGLITMLL VAIFWVWYQR TYAYSHGMDS MEPEFEKVWM GLWRVHMIVM PIFALITWGW IWKTRDTNLD NLDPKLEVK RYFYWMMWLG VYLFGVYWGG SFFTEQDASW HQVIIRDTSF TPSHVVVFYG SFPMYIVCGV ASYLYAMTRL P LYSRGTSF PLVMAIAGPL MILPNVGLNE WGHAFWFMEE LFSAPLHWGF VILGWSGLFA GGIAAQIITR YSNLTDVVWN GQ SKVILNN RIVP

UniProtKB: UNIPROTKB: A0A1G4ZCX6

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Macromolecule #2: Ammonia monooxygenase subunit A

MacromoleculeName: Ammonia monooxygenase subunit A / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Nitrosospira briensis (bacteria)
Molecular weightTheoretical: 31.28876 KDa
Recombinant expressionOrganism: Nitrosospira briensis (bacteria)
SequenceString: SRTDEILKAA KMPPEAVKMS RMIDAVYFPI LCILLVGTYH MHFMLLAGDW DFWLDWKDRQ WWPVVTPIVG ITYCAAIMYY LWVNYRLPF GATLCIVCLL TGEWLTRFWG FYWWSHYPIS FVFPSTMIPG ALVMDTVMLL TRNWMITALV GGGAFGLLFY P GNWTIFGP ...String:
SRTDEILKAA KMPPEAVKMS RMIDAVYFPI LCILLVGTYH MHFMLLAGDW DFWLDWKDRQ WWPVVTPIVG ITYCAAIMYY LWVNYRLPF GATLCIVCLL TGEWLTRFWG FYWWSHYPIS FVFPSTMIPG ALVMDTVMLL TRNWMITALV GGGAFGLLFY P GNWTIFGP THLPLVAEGV LLSVADYTGF LYVRTGTPEY VRLIEQGSLR TFGGHTTVIA SFFAAFVSML MFCVWWYFGK LY CTAFFYV KGARGRVTMK NDVTAFGEEG FPEGIK

UniProtKB: Ammonia monooxygenase subunit A

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Macromolecule #3: Ammonia monooxygenase subunit B

MacromoleculeName: Ammonia monooxygenase subunit B / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Nitrosospira briensis (bacteria)
Molecular weightTheoretical: 42.675074 KDa
Recombinant expressionOrganism: Nitrosospira briensis (bacteria)
SequenceString: HGERSQEPFL RMRTIQWYDM KWGPETTKVN DFASMTGKFH LAEDWPRAVG KPGRAFFNVG SPSPVFVRLS TKLNGEPTMI SGPLEIGRD YAFEVKLKAR IPGRHHMHAM VNIKDAGPIA GPAAWMNITG SWDDFTNPIK LLTGETIDTE TFNFSNGIFW H ALWMALGI ...String:
HGERSQEPFL RMRTIQWYDM KWGPETTKVN DFASMTGKFH LAEDWPRAVG KPGRAFFNVG SPSPVFVRLS TKLNGEPTMI SGPLEIGRD YAFEVKLKAR IPGRHHMHAM VNIKDAGPIA GPAAWMNITG SWDDFTNPIK LLTGETIDTE TFNFSNGIFW H ALWMALGI FWIGYYVARP MFLPRSRVLL AYGDELLIDP MDKKVAWVVL ILTLGIVWGG YRYTENKHPY TVPIQAGESK VA PLPVKPN PIAIKVTHAN YDVPGRALRV TMMVTNSGDS AYRIGEFTTA GVRFINKVGL KHMDRGYPKE LVATGLSFDN DTP IQPGET REVKMEAKDA LWEVQRLMAL LGDPESRFGG LLMTWNDAGD RNINSIAGAV IPVFT

UniProtKB: Ammonia monooxygenase subunit B

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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

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Macromolecule #5: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-...

MacromoleculeName: (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
type: ligand / ID: 5 / Number of copies: 3 / Formula: 6PL
Molecular weightTheoretical: 763.1 Da
Chemical component information

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 / phospholipid*YM

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 848 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.2
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 212075
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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