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- PDB-10zl: CryoEM structure of Gluconacetobacter diazotrophicus MoFeP (C2 sy... -

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Basic information

Entry
Database: PDB / ID: 10zl
TitleCryoEM structure of Gluconacetobacter diazotrophicus MoFeP (C2 symmetry)
Components(Nitrogenase MoFe protein ...) x 2
KeywordsOXIDOREDUCTASE / Nitrogenase / MoFeP / Gluconacetobacter diazotrophicus / molybdenum-iron protein
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / nitrogenase activity / iron-sulfur cluster binding / ATP binding / metal ion binding
Similarity search - Function
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
Similarity search - Domain/homology
FE(8)-S(7) CLUSTER / : / 3-HYDROXY-3-CARBOXY-ADIPIC ACID / Chem-ICS / Nitrogenase protein alpha chain / Nitrogenase molybdenum-iron protein beta chain
Similarity search - Component
Biological speciesGluconacetobacter diazotrophicus PA1 5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.15 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
B: Nitrogenase MoFe protein beta subunit NifK
D: Nitrogenase MoFe protein beta subunit NifK
A: Nitrogenase MoFe protein alpha subunit NifD
C: Nitrogenase MoFe protein alpha subunit NifD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,82112
Polymers226,3804
Non-polymers3,4418
Water00
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 MoFe protein ... , 2 types, 4 molecules BDAC

#1: Protein Nitrogenase MoFe protein beta subunit NifK / Nitrogenase molybdenum-iron protein beta chain


Mass: 57094.352 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Gluconacetobacter diazotrophicus PA1 5 (bacteria)
References: UniProt: A9H5W8, nitrogenase
#2: Protein Nitrogenase MoFe protein alpha subunit NifD


Mass: 56095.453 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Gluconacetobacter diazotrophicus PA1 5 (bacteria)
References: UniProt: A9H5W5, nitrogenase

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Non-polymers , 4 types, 8 molecules

#3: 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
#4: 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
#5: 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
#6: 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

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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: Heterotetrameric MoFeP from Gluconacetobacter diazotrophicus
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.23 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
SpecimenConc.: 6.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Homogeneous Gd-MoFeP purified from a gel filtration column(SEC).
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: 130000 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: 2371
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: 674977
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 211731 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementHighest resolution: 2.15 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01516235
ELECTRON MICROSCOPYf_angle_d1.03822441
ELECTRON MICROSCOPYf_dihedral_angle_d10.7622289
ELECTRON MICROSCOPYf_chiral_restr0.8492311
ELECTRON MICROSCOPYf_plane_restr0.0052839

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