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- EMDB-75568: CryoEM structure of heterologous nitrogenase complex (2:1 G. diaz... -

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Entry
Database: EMDB / ID: EMD-75568
TitleCryoEM structure of heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx (C2 symmetry)
Map data2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) sharp map
Sample
  • Complex: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx
    • Protein or peptide: x 3 types
  • Ligand: x 9 types
KeywordsNitrogenase / Nitrogenase complex / Heterologous Nitrogenase complex / Gluconacetobacter diazotrophicus / Azotobacter vinelandii / molybdenum-iron protein / Iron protein / MoFeP / FeP / FeP-MoFeP / Oxidoreductase
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / nitrogenase activity / iron-sulfur cluster binding / 4 iron, 4 sulfur cluster binding / ATP binding / metal ion binding
Similarity search - Function
Nitrogenase iron protein NifH / NifH/frxC family / NifH/chlL conserved site / 4Fe-4S iron sulfur cluster binding proteins, NifH/frxC family / NifH/frxC family signature 2. / NifH/frxC family signature 1. / NIFH_FRXC family profile. / Nitrogenase molybdenum-iron protein beta chain, N-terminal / Domain of unknown function (DUF3364) / Nitrogenase molybdenum-iron protein alpha chain ...Nitrogenase iron protein NifH / NifH/frxC family / NifH/chlL conserved site / 4Fe-4S iron sulfur cluster binding proteins, NifH/frxC family / NifH/frxC family signature 2. / NifH/frxC family signature 1. / NIFH_FRXC family profile. / Nitrogenase molybdenum-iron protein beta chain, N-terminal / Domain of unknown function (DUF3364) / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain / Nitrogenase component 1, alpha chain / Nitrogenase component 1, conserved site / Nitrogenases component 1 alpha and beta subunits signature 2. / Nitrogenases component 1 alpha and beta subunits signature 1. / : / Nitrogenase/oxidoreductase, component 1 / Nitrogenase component 1 type Oxidoreductase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nitrogenase molybdenum-iron protein beta chain / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase iron protein
Similarity search - Component
Biological speciesGluconacetobacter diazotrophicus PA1 5 (bacteria) / Azotobacter vinelandii DJ (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.22 Å
AuthorsLi Y / Narehood SM / Cook BD / McGuire KL / Tezcan FA / Herzik Jr MA
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM148607 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM138206 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10OD032471 United States
CitationJournal: Biochemistry / Year: 2026
Title: Structural and Functional Characterization of Heterologous Nitrogenase Complexes.
Authors: Yizhou Li / Sarah M Narehood / Brian D Cook / Kelly L McGuire / Mark A Herzik / F Akif Tezcan /
Abstract: Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum-iron protein (MoFeP) ...Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum-iron protein (MoFeP) and the ATP-dependent reductase iron protein (FeP). Although Mo-nitrogenases are widespread across bacteria and archaea and appear to share conserved mechanistic and structural features, FeP and MoFeP show considerable sequence variability across diazotrophs. This raises questions about the conservation of chemomechanical mechanisms coupling FeP-dependent ATP hydrolysis and electron transfer to MoFeP, and about the functional compatibility of nitrogenase components from divergent species. Previous studies showed that some heterologous FeP-MoFeP pairs can functionally complement each other, whereas other pairs lack catalytic activity, but the absence of structural information on such heterologous pairs has limited mechanistic understanding. To this end, we investigated the functional and structural compatibility of FeP and MoFeP from () and (), two phylogenetically and ecologically distinct species. Building on our prior work with -nitrogenase and recently developed cryogenic electron microscopy (cryoEM) protocols, we determined the ADP·BeF-trapped structure of the homologous FeP-MoFeP complex and showed that it adopted the same geometry as its counterpart. Activity measurements showed that heterologous combinations retained 60-80% of homologous catalytic activities despite 30-50% sequence divergence in FeP and MoFeP. High-resolution cryoEM structures of FeP-MoFeP and FeP-MoFeP corroborated these activities and revealed that functional complementation tolerates substantial sequence variation when the core structural elements supporting ATP binding/hydrolysis, protein-protein interaction, electron transfer, and substrate reduction are conserved.
History
DepositionFeb 12, 2026-
Header (metadata) releaseJul 15, 2026-
Map releaseJul 15, 2026-
UpdateJul 15, 2026-
Current statusJul 15, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_75568.png

Downloads & links

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Map

FileDownload / File: emd_75568.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) sharp map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 448 pix.
= 329.28 Å		0.74 Å/pix.
x 448 pix.
= 329.28 Å		0.74 Å/pix.
x 448 pix.
= 329.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.735 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.12640037 - 0.25243205
Average (Standard dev.)0.00013538761 (±0.007060396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 329.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_75568_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) map

Fileemd_75568_additional_1.map
Annotation2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) half map

Fileemd_75568_half_map_1.map
Annotation2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) half map

Fileemd_75568_half_map_2.map
Annotation2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP (C2 symmetry) half 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 : Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vi...

EntireName: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx
Components
  • Complex: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
    • Protein or peptide: Nitrogenase iron protein
  • Ligand: 3-HYDROXY-3-CARBOXY-ADIPIC ACID
  • Ligand: iron-sulfur-molybdenum cluster with interstitial carbon
  • Ligand: FE (III) ION
  • Ligand: FE(8)-S(7) CLUSTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: water

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Supramolecule #1: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vi...

SupramoleculeName: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Gluconacetobacter diazotrophicus PA1 5 (bacteria)
Molecular weightTheoretical: 357 KDa

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Macromolecule #1: Nitrogenase molybdenum-iron protein alpha chain

MacromoleculeName: Nitrogenase molybdenum-iron protein alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 55.363043 KDa
SequenceString: MTGMSREEVE SLIQEVLEVY PEKARKDRNK HLAVNDPAVT QSKKCIISNK KSQPGLMTIR GCAYAGSKGV VWGPIKDMIH ISHGPVGCG QYSRAGRRNY YIGTTGVNAF VTMNFTSDFQ EKDIVFGGDK KLAKLIDEVE TLFPLNKGIS VQSECPIGLI G DDIESVSK ...String:
MTGMSREEVE SLIQEVLEVY PEKARKDRNK HLAVNDPAVT QSKKCIISNK KSQPGLMTIR GCAYAGSKGV VWGPIKDMIH ISHGPVGCG QYSRAGRRNY YIGTTGVNAF VTMNFTSDFQ EKDIVFGGDK KLAKLIDEVE TLFPLNKGIS VQSECPIGLI G DDIESVSK VKGAELSKTI VPVRCEGFRG VSQSLGHHIA NDAVRDWVLG KRDEDTTFAS TPYDVAIIGD YNIGGDAWSS RI LLEEMGL RCVAQWSGDG SISEIELTPK VKLNLVHCYR SMNYISRHME EKYGIPWMEY NFFGPTKTIE SLRAIAAKFD ESI QKKCEE VIAKYKPEWE AVVAKYRPRL EGKRVMLYIG GLRPRHVIGA YEDLGMEVVG TGYEFAHNDD YDRTMKEMGD STLL YDDVT GYEFEEFVKR IKPDLIGSGI KEKFIFQKMG IPFREMHSWD YSGPYHGFDG FAIFARDMDM TLNNPCWKKL QAPWE ASEG AEKVAASA

UniProtKB: Nitrogenase molybdenum-iron protein alpha chain

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Macromolecule #2: Nitrogenase molybdenum-iron protein beta chain

MacromoleculeName: Nitrogenase molybdenum-iron protein beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Molecular weightTheoretical: 59.535879 KDa
SequenceString: MSQQVDKIKA SYPLFLDQDY KDMLAKKRDG FEEKYPQDKI DEVFQWTTTK EYQELNFQRE ALTVNPAKAC QPLGAVLCAL GFEKTMPYV HGSQGCVAYF RSYFNRHFRE PVSCVSDSMT EDAAVFGGQQ NMKDGLQNCK ATYKPDMIAV STTCMAEVIG D DLNAFINN ...String:
MSQQVDKIKA SYPLFLDQDY KDMLAKKRDG FEEKYPQDKI DEVFQWTTTK EYQELNFQRE ALTVNPAKAC QPLGAVLCAL GFEKTMPYV HGSQGCVAYF RSYFNRHFRE PVSCVSDSMT EDAAVFGGQQ NMKDGLQNCK ATYKPDMIAV STTCMAEVIG D DLNAFINN SKKEGFIPDE FPVPFAHTPS FVGSHVTGWD NMFEGIARYF TLKSMDDKVV GSNKKINIVP GFETYLGNFR VI KRMLSEM GVGYSLLSDP EEVLDTPADG QFRMYAGGTT QEEMKDAPNA LNTVLLQPWH LEKTKKFVEG TWKHEVPKLN IPM GLDWTD EFLMKVSEIS GQPIPASLTK ERGRLVDMMT DSHTWLHGKR FALWGDPDFV MGLVKFLLEL GCEPVHILCH NGNK RWKKA VDAILAASPY GKNATVYIGK DLWHLRSLVF TDKPDFMIGN SYGKFIQRDT LHKGKEFEVP LIRIGFPIFD RHHLH RSTT LGYEGAMQIL TTLVNSILER LDEETRGMQA TDYNHDLVR

UniProtKB: Nitrogenase molybdenum-iron protein beta chain

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Macromolecule #3: Nitrogenase iron protein

MacromoleculeName: Nitrogenase iron protein / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO / EC number: nitrogenase
Source (natural)Organism: Gluconacetobacter diazotrophicus PA1 5 (bacteria)
Molecular weightTheoretical: 31.907424 KDa
SequenceString: MSKLRQIAFY GKGGIGKSTT SQNTLAALVE MGQKILIVGC DPKADSTRLI LNAKAQDTVL SLAAEAGSVE DLELEDVLKI GYKGIKCVE SGGPEPGVGC AGRGVITSIN FLEENGAYDD VDYVSYDVLG DVVCGGFAMP IRENKAQEIY IVMSGEMMAL Y AANNIAKG ...String:
MSKLRQIAFY GKGGIGKSTT SQNTLAALVE MGQKILIVGC DPKADSTRLI LNAKAQDTVL SLAAEAGSVE DLELEDVLKI GYKGIKCVE SGGPEPGVGC AGRGVITSIN FLEENGAYDD VDYVSYDVLG DVVCGGFAMP IRENKAQEIY IVMSGEMMAL Y AANNIAKG ILKYAHSGGV RLGGLICNER QTDREYDLAD ALAKRLNSKL VHFVPRANIV QHAELRKQTV IEYAPDSAQA GE YRTLAQK IHANSGQGTV PTPITMEELE DMLLEFGIMK TDEQALAELA AKEAKAAAAL A

UniProtKB: Nitrogenase iron protein

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Macromolecule #4: 3-HYDROXY-3-CARBOXY-ADIPIC ACID

MacromoleculeName: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 4 / Number of copies: 2 / Formula: HCA
Molecular weightTheoretical: 206.15 Da
Chemical component information

ChemComp-HCA:
3-HYDROXY-3-CARBOXY-ADIPIC ACID

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Macromolecule #5: iron-sulfur-molybdenum cluster with interstitial carbon

MacromoleculeName: iron-sulfur-molybdenum cluster with interstitial carbon
type: ligand / ID: 5 / Number of copies: 2 / Formula: ICS
Molecular weightTheoretical: 787.451 Da
Chemical component information

ChemComp-ICE:
iron-sulfur-molybdenum cluster with interstitial carbon

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Macromolecule #6: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #7: FE(8)-S(7) CLUSTER

MacromoleculeName: FE(8)-S(7) CLUSTER / type: ligand / ID: 7 / Number of copies: 2 / Formula: CLF
Molecular weightTheoretical: 671.215 Da
Chemical component information

ChemComp-CLF:
FE(8)-S(7) CLUSTER

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #9: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 9 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #10: BERYLLIUM

MacromoleculeName: BERYLLIUM / type: ligand / ID: 10 / Number of copies: 4 / Formula: 0BE
Molecular weightTheoretical: 9.012 Da
Chemical component information

ChemComp-0BE:
BERYLLIUM

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Macromolecule #11: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 11 / Number of copies: 2 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #12: water

MacromoleculeName: water / type: ligand / ID: 12 / Number of copies: 415 / 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 stateparticle

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Sample preparation

Concentration5.22 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
25.0 mMNaClSodium chloride
10.0 mMNa2S2O4Sodium dithionite
5.0 mMMgCl2Magnesium chloride
5.0 mMC10H16N5O13P3Adenosine triphosphate
5.0 mMBeSO4Beryllium sulfate
25.0 mMNaFSodium fluoride

Details: Solutions were prepared, filtered, and degassed to Ar immediately prior to the experiment.
GridModel: Quantifoil Active R1.2/0.8 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 298 K / Instrument: SPT LABTECH CHAMELEON
Details: Samples were covered by Al's oil and frozen with the SPT Labtech chameleon..
DetailsHeterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 3703 / Average exposure time: 5.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 727134
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

26757

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 35330
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-10zm:
CryoEM structure of heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx (C2 symmetry)

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