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- PDB-10zm: CryoEM structure of heterologous nitrogenase complex (2:1 G. diaz... -

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Basic information

Entry
Database: PDB / ID: 10zm
TitleCryoEM structure of heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx (C2 symmetry)
Components
  • (Nitrogenase molybdenum-iron protein ...) x 2
  • Nitrogenase iron protein
KeywordsOXIDOREDUCTASE / Nitrogenase / Nitrogenase complex / Heterologous Nitrogenase complex / Gluconacetobacter diazotrophicus / Azotobacter vinelandii / molybdenum-iron protein / Iron protein / MoFeP / FeP / FeP-MoFeP
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
BERYLLIUM / ADENOSINE-5'-DIPHOSPHATE / FE(8)-S(7) CLUSTER / : / 3-HYDROXY-3-CARBOXY-ADIPIC ACID / Chem-ICS / IRON/SULFUR CLUSTER / Nitrogenase molybdenum-iron protein beta chain / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase iron protein
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
Gluconacetobacter diazotrophicus PA1 5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.22 Å
AuthorsLi, Y. / Narehood, S.M. / Cook, B.D. / McGuire, K.L. / Tezcan, F.A. / Herzik Jr., M.A.
Funding support United States, 3items
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, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 15, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 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

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Assembly

Deposited unit
C: Nitrogenase molybdenum-iron protein alpha chain
D: Nitrogenase molybdenum-iron protein beta chain
A: Nitrogenase molybdenum-iron protein alpha chain
B: Nitrogenase molybdenum-iron protein beta chain
E: Nitrogenase iron protein
F: Nitrogenase iron protein
H: Nitrogenase iron protein
G: Nitrogenase iron protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)363,41430
Polymers357,4288
Non-polymers5,98722
Water7,476415
1


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

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Components

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Nitrogenase molybdenum-iron protein ... , 2 types, 4 molecules CADB

#1: Protein Nitrogenase molybdenum-iron protein alpha chain / Dinitrogenase / Nitrogenase component I


Mass: 55363.043 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii DJ (bacteria) / References: UniProt: P07328, nitrogenase
#2: Protein Nitrogenase molybdenum-iron protein beta chain / Dinitrogenase


Mass: 59535.879 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DGZ8, nitrogenase

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Protein , 1 types, 4 molecules EFHG

#3: Protein
Nitrogenase iron protein / Nitrogenase Fe protein / Nitrogenase component II / Nitrogenase reductase


Mass: 31907.424 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Source: (natural) Gluconacetobacter diazotrophicus PA1 5 (bacteria)
References: UniProt: Q9ZIE4, nitrogenase

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Non-polymers , 9 types, 437 molecules

#4: Chemical ChemComp-HCA / 3-HYDROXY-3-CARBOXY-ADIPIC ACID


Mass: 206.150 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H10O7 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ICS / iron-sulfur-molybdenum cluster with interstitial carbon


Mass: 787.451 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CFe7MoS9 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-CLF / FE(8)-S(7) CLUSTER


Mass: 671.215 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe8S7 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#10: Chemical
ChemComp-0BE / BERYLLIUM


Mass: 9.012 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Be
#11: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 415 / 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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.357 MDa / Experimental value: NO
Source (natural)Organism: Gluconacetobacter diazotrophicus PA1 5 (bacteria)
Buffer solutionpH: 8
Details: Solutions were prepared, filtered, and degassed to Ar immediately prior to the experiment.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
225 mMSodium chlorideNaCl1
310 mMSodium dithioniteNa2S2O41
45 mMMagnesium chlorideMgCl21
55 mMAdenosine triphosphateC10H16N5O13P31
65 mMBeryllium sulfateBeSO41
725 mMSodium fluorideNaF1
SpecimenConc.: 5.22 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Heterologous nitrogenase complex (2:1 G. diazotrophicus-FeP:A. vinelandii-MoFeP) inhibited by BeFx
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil Active R1.2/0.8
VitrificationInstrument: SPT LABTECH CHAMELEON / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 298 K
Details: Samples were covered by Al's oil and frozen with the SPT Labtech chameleon.

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3703
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU2image acquisition
4cryoSPARCCTF correction
9PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 727134
3D reconstructionResolution: 2.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35330 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementHighest resolution: 2.22 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01224998
ELECTRON MICROSCOPYf_angle_d0.99734278
ELECTRON MICROSCOPYf_dihedral_angle_d10.1723514
ELECTRON MICROSCOPYf_chiral_restr0.6753676
ELECTRON MICROSCOPYf_plane_restr0.0064342

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