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- EMDB-52557: CryoEM structure of holo-GmNifEN -

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Basic information

Entry
Database: EMDB / ID: EMD-52557
TitleCryoEM structure of holo-GmNifEN
Map dataCryoEM structure of holo-GmNifEN sharped with EMready
Sample
  • Complex: nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN
    • Protein or peptide: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FeFe cofactor
KeywordsNITROGENASE COFACTOR MATURATION / PROTEIN BINDING / Metalloenzyme / Iron-Sulfur Cluster / METAL BINDING PROTEIN
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
Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
Similarity search - Component
Biological speciesGeobacter metallireducens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsPaya Tormo L / Nguyen TQ / Fyfe C / Basbous H / Dobrzynska K / Echavarri-Erasun C / Martin L / Caserta G / Legrand P / Thorn A ...Paya Tormo L / Nguyen TQ / Fyfe C / Basbous H / Dobrzynska K / Echavarri-Erasun C / Martin L / Caserta G / Legrand P / Thorn A / Amara P / Schoehn G / Cherrier MV / Rubio LM / Nicolet Y
Funding support France, 3 items
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, 2025-
Header (metadata) releaseNov 5, 2025-
Map releaseNov 5, 2025-
UpdateNov 26, 2025-
Current statusNov 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52557.png

Downloads & links

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Map

FileDownload / File: emd_52557.map.gz / Format: CCP4 / Size: 56.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of holo-GmNifEN sharped with EMready
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 246 pix.
= 280.44 Å		1.14 Å/pix.
x 246 pix.
= 280.44 Å		1.14 Å/pix.
x 246 pix.
= 280.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-0.74619305 - 21.309170000000002
Average (Standard dev.)-0.010523333 (±0.63368565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions246246246
Spacing246246246
CellA=B=C: 280.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: CryoEM structure of holo-GmNifEN unsharped

Fileemd_52557_additional_1.map
AnnotationCryoEM structure of holo-GmNifEN unsharped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: CryoEM structure of holo-GmNifEN sharped

Fileemd_52557_additional_2.map
AnnotationCryoEM structure of holo-GmNifEN sharped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM structure of holo-GmNifEN

Fileemd_52557_half_map_1.map
AnnotationCryoEM structure of holo-GmNifEN
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM structure of holo-GmNifEN

Fileemd_52557_half_map_2.map
AnnotationCryoEM structure of holo-GmNifEN
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN

EntireName: nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN
Components
  • Complex: nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN
    • Protein or peptide: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FeFe cofactor

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Supramolecule #1: nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN

SupramoleculeName: nitrogenase iron-molybdenum cofactor biosynthesis protein NifEN
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Geobacter metallireducens (bacteria)
Molecular weightTheoretical: 199.774 KDa

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Macromolecule #1: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE

MacromoleculeName: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Geobacter metallireducens (bacteria) / Strain: strain ATCC 53774 / DSM 7210 / GS-15
Molecular weightTheoretical: 100.135594 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKPDYYDVS ECETHEKGAP KFCKKSEPGE GAERSCAYDG ARVVLMPITD VIHLVHGPIA CAGNSWDNRG ARSSDSQLYR RGFTTEMLE NDVVFGGEKK LYRAILELAE RYEGQAKAMF VYATCVTAMT GDDVEAVCAA AGKKVAIPLI PVNTPGFIGD K NIGNRLAG ...String:
MAKPDYYDVS ECETHEKGAP KFCKKSEPGE GAERSCAYDG ARVVLMPITD VIHLVHGPIA CAGNSWDNRG ARSSDSQLYR RGFTTEMLE NDVVFGGEKK LYRAILELAE RYEGQAKAMF VYATCVTAMT GDDVEAVCAA AGKKVAIPLI PVNTPGFIGD K NIGNRLAG EVLFKHVIGT AEPPVLGEYP INLIGEYNIA GDLWGMLPLF ERLGIQVLSC FSGDATFEEL RYAHRAKLNI II CSKSLTN LARKMQKNYG MPYLEESFYG MTDTAKALRD IARELDDAVG GLEKRIMQDR VEKLLEEEEA TCRERLAPYR ARL EGKRSV LFTGGVKTWS MVNALRELGV EILAAGTQNS TLEDFYRMKA LMHQDARIIE DTSSAGLLQV MYDKMPDLIV AGGK TKFLA LKTKTPFLDI NHGRSHPYAG YEGMVTFAKQ LDLTVNNPIW PVLNAKAPWE KTEEELTAAV ALAAGHARAC LDEDL KDST VKVPAKNATV NPQKNSPALG ATLAYLGIDQ MLALLHGAQG CSTFIRLQLS RHFKEPVALN STAMSEDTAI FGGWEN LKK GLKKVIEKFS PEVVGVMTSG LTETMGDDVR SAIVHFRQEY PEHDGVPVVW ASTPDYCGSL QEGYAATVEA IVRSVPE PG ETIPGQVTVL PGAHLTPADV EEVRELCEAF GLDPIIVPDI ANALDGHIDE TVSPLSTGGV SMARIRQAGQ SAATLFIG D SLAKAAEAMT ERCGMPSYGF TSLTGLAQVD RFMETLAAIA GRPIPEKFRR WRSRLMDAMV DSHYQFGLKK VTVALEGDN LKTLVNFLAG MGCEIQAAIA ATRVRGLDGL PARDIFVGDL EDLETAARGS DLIVANSNGR QAAAKLGIKA HLRAGLPVFD RLGAHQKMW VGYRGTMNLL FETANLFQAN AGEGQKLAHN

UniProtKB: Nitrogenase iron-molybdenum cofactor biosynthesis protein NifE

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Macromolecule #2: IRON/SULFUR CLUSTER

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

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #3: FeFe cofactor

MacromoleculeName: FeFe cofactor / type: ligand / ID: 3 / Number of copies: 2 / Formula: S5Q
Molecular weightTheoretical: 747.356 Da
Chemical component information

ChemComp-S5Q:
FeFe cofactor

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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

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationName
50.0 mMTris-HCl
2.0 mMDTH
500.0 mMNaCl
GridModel: Quantifoil R2/1 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 30 mA
VitrificationCryogen name: PROPANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-40 / Number grids imaged: 1 / Number real images: 2758 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 36000

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Image processing

Particle selectionNumber selected: 2672130
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold models of NifEN dimer
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 178282
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: PHENIX (ver. 1.21.1_5286:)
DetailsAlphaFold Model of gmNifEN
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9i0g:
CryoEM structure of holo-GmNifEN

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