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- EMDB-71994: Cryo-EM structure of NapA, the periplasmic nitrate reductase from... -

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Basic information

Entry
Database: EMDB / ID: EMD-71994
TitleCryo-EM structure of NapA, the periplasmic nitrate reductase from Campylobacter jejuni
Map data
Sample
  • Complex: Structure of NapA:Moco[4Fe4S]
    • Protein or peptide: Periplasmic nitrate reductase
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MOLYBDENUM ATOM
  • Ligand: 2-AMINO-5,6-DIMERCAPTO-7-METHYL-3,7,8A,9-TETRAHYDRO-8-OXA-1,3,9,10-TETRAAZA-ANTHRACEN-4-ONE GUANOSINE DINUCLEOTIDE
KeywordsPeriplasmic nitrate reductase / molybdenum enzymes / cryo-EM / MEMBRANE PROTEIN / METAL BINDING PROTEIN / OXIDOREDUCTASE
Function / homology
Function and homology information


nitrate reductase (cytochrome) / nitrate reductase (cytochrome) activity / nitrate reductase complex / Mo-molybdopterin cofactor biosynthetic process / molybdenum ion binding / molybdopterin cofactor binding / cellular respiration / nitrate assimilation / 4 iron, 4 sulfur cluster binding / periplasmic space ...nitrate reductase (cytochrome) / nitrate reductase (cytochrome) activity / nitrate reductase complex / Mo-molybdopterin cofactor biosynthetic process / molybdenum ion binding / molybdopterin cofactor binding / cellular respiration / nitrate assimilation / 4 iron, 4 sulfur cluster binding / periplasmic space / electron transfer activity / iron ion binding / membrane
Similarity search - Function
Periplasmic nitrate reductase, large subunit / Nitrate reductase NapA-like, molybdopterin-binding domain / Molybdopterin oxidoreductase, molybdopterin cofactor binding site / Prokaryotic molybdopterin oxidoreductases signature 1. / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin dinucleotide-binding domain / Molydopterin dinucleotide binding domain / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / : ...Periplasmic nitrate reductase, large subunit / Nitrate reductase NapA-like, molybdopterin-binding domain / Molybdopterin oxidoreductase, molybdopterin cofactor binding site / Prokaryotic molybdopterin oxidoreductases signature 1. / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin dinucleotide-binding domain / Molydopterin dinucleotide binding domain / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / : / Aspartate decarboxylase-like domain superfamily / Molybdopterin oxidoreductase, 4Fe-4S domain / Prokaryotic molybdopterin oxidoreductases 4Fe-4S domain profile. / Molybdopterin oxidoreductase / Molybdopterin oxidoreductase
Similarity search - Domain/homology
Periplasmic nitrate reductase
Similarity search - Component
Biological speciesCampylobacter jejuni (Campylobacter)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsThach T / Subramanian R
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biol Chem / Year: 2025
Title: Structure and substrate promiscuity of Campylobacter jejuni periplasmic nitrate reductase (Nap) and phylogenetic analysis of Nap homologs.
Authors: Nitai C Giri / Trung Thach / KanagaVijayan Dhanabalan / Mintare Cesiunaite / Manohar Radhakrishnan / Lahiru Wedasingha / Nicholas Manicke / Michael Wells / Maciej Szaleniec / Ramaswamy ...Authors: Nitai C Giri / Trung Thach / KanagaVijayan Dhanabalan / Mintare Cesiunaite / Manohar Radhakrishnan / Lahiru Wedasingha / Nicholas Manicke / Michael Wells / Maciej Szaleniec / Ramaswamy Subramanian / Partha Basu /
Abstract: Periplasmic nitrate reductase NapA is a member of the DMSO reductase (DMSOR) superfamily, which catalyzes the reduction of nitrate to nitrite. Campylobacter jejuni NapA (CjNapA) is notably larger ...Periplasmic nitrate reductase NapA is a member of the DMSO reductase (DMSOR) superfamily, which catalyzes the reduction of nitrate to nitrite. Campylobacter jejuni NapA (CjNapA) is notably larger compared to other structurally characterized NapA. Herein, we present the cryo-EM structure of CjNapA, the first of its kind from any ε-proteobacteria, revealing three lysine-rich insertions that could affect the substrate channel, potentially enhancing the affinity towards nitrate and other anionic substrates. Here, we report that wild-type CjNapA and NapA-C176D variants can reduce chlorate, perchlorate, and nitrate. However, the perchlorate and chlorate reductions by the CjNapA C176D variant are considerably slower, even though the perchlorate reductase has an Asp coordination to Mo. Molecular Dynamics (MD) simulations were performed to investigate the impact of the C176D mutation on substrate affinity and protein flexibility. Structural and kinetic comparisons with perchlorate reductase support evolutionary tuning for a desired function. Finally, structural comparisons with other structurally characterized NapAs also suggest the role of proximal pterin in CjNapA in electron transfer to the Mo center.
History
DepositionAug 6, 2025-
Header (metadata) releaseJun 17, 2026-
Map releaseJun 17, 2026-
UpdateJun 17, 2026-
Current statusJun 17, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71994.png

Downloads & links

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Map

FileDownload / File: emd_71994.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 256 pix.
= 210.432 Å		0.82 Å/pix.
x 256 pix.
= 210.432 Å		0.82 Å/pix.
x 256 pix.
= 210.432 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-1.2578554 - 2.3418212
Average (Standard dev.)0.0017544227 (±0.044261277)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_71994_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_71994_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Structure of NapA:Moco[4Fe4S]

EntireName: Structure of NapA:Moco[4Fe4S]
Components
  • Complex: Structure of NapA:Moco[4Fe4S]
    • Protein or peptide: Periplasmic nitrate reductase
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MOLYBDENUM ATOM
  • Ligand: 2-AMINO-5,6-DIMERCAPTO-7-METHYL-3,7,8A,9-TETRAHYDRO-8-OXA-1,3,9,10-TETRAAZA-ANTHRACEN-4-ONE GUANOSINE DINUCLEOTIDE

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Supramolecule #1: Structure of NapA:Moco[4Fe4S]

SupramoleculeName: Structure of NapA:Moco[4Fe4S] / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
Molecular weightTheoretical: 84 KDa

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Macromolecule #1: Periplasmic nitrate reductase

MacromoleculeName: Periplasmic nitrate reductase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: nitrate reductase (cytochrome)
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
Molecular weightTheoretical: 105.092828 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNRRDFIKNT AIASAASVAG LSVPSSMLGA QEEDWKWDKA VCRFCGTGCG IMIARKDGKI VATKGDPAAP VNRGLNCIKG YFNAKIMYG EDRLVMPLLR MNEKGEFDKK GKFQQVSWQR AFDEMEKQFK KAYNELGVTG IGIFGSGQYT IQEGYAALKL A KAGFRTNN ...String:
MNRRDFIKNT AIASAASVAG LSVPSSMLGA QEEDWKWDKA VCRFCGTGCG IMIARKDGKI VATKGDPAAP VNRGLNCIKG YFNAKIMYG EDRLVMPLLR MNEKGEFDKK GKFQQVSWQR AFDEMEKQFK KAYNELGVTG IGIFGSGQYT IQEGYAALKL A KAGFRTNN IDPNARHCMA SAVVGFMQTF GVDEPSGCYD DIELTDTIIT WGANMAEMHP ILWSRVSDRK LSNLDKVKVV NL STFSNRT SNIADIEIIF KPNTDLAIWN YIAREIVYNH PEAMDMKFIK DHCVFATGYA DIGYGMRNNP NHPKFKESEK DTV EKENVI TLDDEEATSL SYLGVKAGDK FEMKHQGVAD KNWEISFDEF KKGLAPYTLE YTARVAKGDD NESLEDFKKK LQEL ANLYI EKNRKVVSFW TMGFNQHTRG SWVNEQAYMV HFLLGKQAKP GSGAFSLTGQ PSACGTAREV GTFSHRLPAD MVVAN PKHR EISEKIWKVP AKTINPKPGS PYLNIMRDLE DGKIKFAWVQ VNNPWQNTAN ANHWIAAARE MDNFIVVSDC YPGISA KVA DLILPSAMIY EKWGAYGNAE RRTQHWKQQV LPVGAAMSDT WQILEFAKRF KLKEVWKEQK VDNKLTLPSV LEEAKAM GY SEDDTLFDVL FANKEAKSFN PNDAIAKGFD NTDVKGDERK IQGSDGKEFT GYGFFVQKYL WEEYRKFGLG HGHDLADF D TYHKVRGLRW PVVNGKETQW RFNTKFDYYA KKAAPNSDFA FYGDFNKMLT NGDLIAPKDE KEHSIKNKAK IFFRPFMKA PERPSKEYPF WLATGRVLEH WHSGTMTMRV PELYRAVPEA LCYMSEKDGE KLGLNQGDLV WVESRRGKVK ARVDMRGRNK PPVGLVYVP WFDENVYINK VTLDATCPLS KQTDFKKCAV KIYKA

UniProtKB: Periplasmic nitrate reductase

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Macromolecule #2: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 2 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #3: MOLYBDENUM ATOM

MacromoleculeName: MOLYBDENUM ATOM / type: ligand / ID: 3 / Number of copies: 1 / Formula: MO
Molecular weightTheoretical: 95.94 Da
Chemical component information

ChemComp-MO:
MOLYBDENUM ATOM

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Macromolecule #4: 2-AMINO-5,6-DIMERCAPTO-7-METHYL-3,7,8A,9-TETRAHYDRO-8-OXA-1,3,9,1...

MacromoleculeName: 2-AMINO-5,6-DIMERCAPTO-7-METHYL-3,7,8A,9-TETRAHYDRO-8-OXA-1,3,9,10-TETRAAZA-ANTHRACEN-4-ONE GUANOSINE DINUCLEOTIDE
type: ligand / ID: 4 / Number of copies: 2 / Formula: MGD
Molecular weightTheoretical: 740.557 Da
Chemical component information

ChemComp-MGD:
2-AMINO-5,6-DIMERCAPTO-7-METHYL-3,7,8A,9-TETRAHYDRO-8-OXA-1,3,9,10-TETRAAZA-ANTHRACEN-4-ONE GUANOSINE DINUCLEOTIDE

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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

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMNaClSodium Chloride
0.5 mMTCEPTris(2-carboxyethyl)phosphine
2.0 %GlycerolGlycerol

Details: 25 mM Hepes 7.0, 100 mM NaCl, 0.5 mM TCEP, 2% glycerol
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: CONTINUOUS / Support film - Film thickness: 11
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: vitrification.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Number grids imaged: 1 / Number real images: 60 / Average exposure time: 1.8 sec. / Average electron dose: 56.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.1 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 200982
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: ab initio construction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 131448
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 50
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Chain ID: A / Chain - Residue range: 1-924 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: The initial model consisted of monomer
Detailsreal refinement was done using Phenix
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9pxt:
Cryo-EM structure of NapA, the periplasmic nitrate reductase from Campylobacter jejuni

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