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- PDB-9i0g: CryoEM structure of holo-GmNifEN -

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Basic information

Entry
Database: PDB / ID: 9i0g
TitleCryoEM structure of holo-GmNifEN
ComponentsNitrogenase iron-molybdenum cofactor biosynthesis protein NifE
KeywordsMETAL BINDING PROTEIN / NITROGENASE COFACTOR MATURATION / PROTEIN BINDING / Metalloenzyme / Iron-Sulfur Cluster
Function / homology
Function and homology information


nitrogenase activity / protein-containing complex assembly
Similarity search - Function
Nitrogenase MoFe cofactor biosynthesis protein NifE / Nitrogenase molybdenum-iron cofactor biosynthesis protein / : / 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
FeFe cofactor / IRON/SULFUR CLUSTER / Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
Similarity search - Component
Biological speciesGeobacter metallireducens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsPaya Tormo, L. / Nguyen, T.Q. / Fyfe, C. / Basbous, H. / Dobrzynska, K. / Echavarri-Erasun, C. / Martin, L. / Caserta, G. / Legrand, P. / Thorn, A. ...Paya Tormo, L. / Nguyen, T.Q. / Fyfe, C. / Basbous, H. / Dobrzynska, K. / Echavarri-Erasun, C. / Martin, L. / Caserta, G. / Legrand, P. / Thorn, A. / Amara, P. / Schoehn, G. / Cherrier, M.V. / Rubio, L.M. / Nicolet, Y.
Funding support France, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-15-IDEX-02 France
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INBS-05-02 France
Grenoble Alliance for Integrated Structural Cell Biology (GRAL)ANR-17-EURE-0003 France
CitationJournal: Nat Chem Biol / Year: 2025
Title: Dynamics driving the precursor in NifEN scaffold during nitrogenase FeMo-cofactor assembly.
Authors: Lucía Payá Tormo / Tu-Quynh Nguyen / Cameron Fyfe / Hind Basbous / Katarzyna Dobrzyńska / Carlos Echavarri-Erasun / Lydie Martin / Giorgio Caserta / Pierre Legrand / Andrea Thorn / ...Authors: Lucía Payá Tormo / Tu-Quynh Nguyen / Cameron Fyfe / Hind Basbous / Katarzyna Dobrzyńska / Carlos Echavarri-Erasun / Lydie Martin / Giorgio Caserta / Pierre Legrand / Andrea Thorn / Patricia Amara / Guy Schoehn / Mickaël V Cherrier / Luis M Rubio / Yvain Nicolet /
Abstract: Nitrogenase catalyzes atmospheric nitrogen fixation, a critical biological process that depends on an intricate organometallic cofactor assembled by a dedicated multiprotein system. Here we uncover ...Nitrogenase catalyzes atmospheric nitrogen fixation, a critical biological process that depends on an intricate organometallic cofactor assembled by a dedicated multiprotein system. Here we uncover the structural basis for the function of NifEN, the scaffold protein that mediates the final stages of cofactor biosynthesis before its incorporation into nitrogenase. High-resolution structural analyses reveal that the cofactor precursor initially binds at a surface docking site before being transferred into a specialized cavity for further maturation. This process involves dynamic structural rearrangements, including coordinated domain motions and partial unfolding, enabling the scaffold to alternate between open and closed states. Additionally, a rear channel extends to the precursor-binding cavity, likely facilitating the entry of the modifying components molybdenum and homocitrate. These findings illuminate the dynamic mechanisms underlying FeMo-cofactor assembly and underscore the functional divergence between NifEN, the biosynthetic scaffold, and NifDK, the catalytic component of nitrogenase.
History
DepositionJan 15, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 5, 2025Provider: repository / Type: Initial release
Revision 1.1Nov 26, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
B: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)202,4696
Polymers200,2712
Non-polymers2,1984
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE / Nitrogenase iron-molybdenum cofactor biosynthesis protein NifN


Mass: 100135.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter metallireducens (bacteria) / Strain: strain ATCC 53774 / DSM 7210 / GS-15 / Gene: nifEN, Gmet_0669 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q39XW3
#2: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4
#3: Chemical ChemComp-S5Q / FeFe cofactor


Mass: 747.356 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CFe8S9
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: nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.199774 MDa / Experimental value: NO
Source (natural)Organism: Geobacter metallireducens (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HCl1
22 mMDTH1
3500 mMNaCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA / Grid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: PROPANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2758
Image scansMovie frames/image: 40 / Used frames/image: 2-40

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Processing

EM software
IDNameVersionCategory
12PHENIX1.21.1_5286:model fitting
18EMReadyother
19PHENIX1.21.1_5286:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2672130
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178282 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: AlphaFold Model of gmNifEN
Atomic model buildingSource name: AlphaFold / Type: in silico model

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