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- EMDB-45923: Azotobacter vinelandii MoFeP (C2 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-45923
TitleAzotobacter vinelandii MoFeP (C2 symmetry)
Map dataSharpened EM map of MoFeP; C2 symmetry
Sample
  • Complex: Azotobacter vinelandii MoFeP (C2 symmetry)
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
  • 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: water
KeywordsNitrogenase / FeMoCo / nitrogen / P-cluster / METAL BINDING PROTEIN
Function / homology
Function and homology information


molybdenum-iron nitrogenase complex / nitrogenase / nitrogenase activity / nitrogen fixation / 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
Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain
Similarity search - Component
Biological speciesAzotobacter vinelandii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsNarehood SM / Cook BD / Srisantitham S / Eng VH / Shiau A / Britt RD / Herzik MA / Tezcan FA
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM148607-02 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM138206 United States
Other privateSearle Scholars
CitationJournal: Nature / Year: 2025
Title: Structural basis for the conformational protection of nitrogenase from O.
Authors: Sarah M Narehood / Brian D Cook / Suppachai Srisantitham / Vanessa H Eng / Angela A Shiau / Kelly L McGuire / R David Britt / Mark A Herzik / F Akif Tezcan /
Abstract: The low reduction potentials required for the reduction of dinitrogen (N) render metal-based nitrogen-fixation catalysts vulnerable to irreversible damage by dioxygen (O). Such O sensitivity ...The low reduction potentials required for the reduction of dinitrogen (N) render metal-based nitrogen-fixation catalysts vulnerable to irreversible damage by dioxygen (O). Such O sensitivity represents a major conundrum for the enzyme nitrogenase, as a large fraction of nitrogen-fixing organisms are either obligate aerobes or closely associated with O-respiring organisms to support the high energy demand of catalytic N reduction. To counter O damage to nitrogenase, diazotrophs use O scavengers, exploit compartmentalization or maintain high respiration rates to minimize intracellular O concentrations. A last line of damage control is provided by the 'conformational protection' mechanism, in which a [2Fe:2S] ferredoxin-family protein termed FeSII (ref. ) is activated under O stress to form an O-resistant complex with the nitrogenase component proteins. Despite previous insights, the molecular basis for the conformational O protection of nitrogenase and the mechanism of FeSII activation are not understood. Here we report the structural characterization of the Azotobacter vinelandii FeSII-nitrogenase complex by cryo-electron microscopy. Our studies reveal a core complex consisting of two molybdenum-iron proteins (MoFePs), two iron proteins (FePs) and one FeSII homodimer, which polymerize into extended filaments. In this three-protein complex, FeSII mediates an extensive network of interactions with MoFeP and FeP to position their iron-sulphur clusters in catalytically inactive but O-protected states. The architecture of the FeSII-nitrogenase complex is confirmed by solution studies, which further indicate that the activation of FeSII involves an oxidation-induced conformational change.
History
DepositionJul 25, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45923.png

Downloads & links

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Map

FileDownload / File: emd_45923.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened EM map of MoFeP; C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 384 pix.
= 282.24 Å		0.74 Å/pix.
x 384 pix.
= 282.24 Å		0.74 Å/pix.
x 384 pix.
= 282.24 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: 0.12
Minimum - Maximum-0.3256291 - 0.6150345
Average (Standard dev.)0.0004999583 (±0.020553079)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 282.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45923_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened EM map of MoFeP; C2 symmetry

Fileemd_45923_additional_1.map
AnnotationUnsharpened EM map of MoFeP; C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of MoFeP; C2 symmetry

Fileemd_45923_half_map_1.map
AnnotationHalf map B of MoFeP; C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of MoFeP; C2 symmetry

Fileemd_45923_half_map_2.map
AnnotationHalf map A of MoFeP; C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Azotobacter vinelandii MoFeP (C2 symmetry)

EntireName: Azotobacter vinelandii MoFeP (C2 symmetry)
Components
  • Complex: Azotobacter vinelandii MoFeP (C2 symmetry)
    • Protein or peptide: Nitrogenase molybdenum-iron protein alpha chain
    • Protein or peptide: Nitrogenase molybdenum-iron protein beta chain
  • 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: water

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Supramolecule #1: Azotobacter vinelandii MoFeP (C2 symmetry)

SupramoleculeName: Azotobacter vinelandii MoFeP (C2 symmetry) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Azotobacter vinelandii (bacteria)
Molecular weightTheoretical: 230 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 (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 (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: 3-HYDROXY-3-CARBOXY-ADIPIC ACID

MacromoleculeName: 3-HYDROXY-3-CARBOXY-ADIPIC ACID / type: ligand / ID: 3 / 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 #4: iron-sulfur-molybdenum cluster with interstitial carbon

MacromoleculeName: iron-sulfur-molybdenum cluster with interstitial carbon
type: ligand / ID: 4 / 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 #5: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #6: FE(8)-S(7) CLUSTER

MacromoleculeName: FE(8)-S(7) CLUSTER / type: ligand / ID: 6 / 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 #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 924 / 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

Concentration2.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
25.0 mMNaClSodium Chloride
5.0 mMNa2S2O4Sodium Dithionite
5.0 mMMgCl2Magnesium chloride
5.0 mMC10H16N5O13P3Adenosine triphosphate
GridModel: Quantifoil / 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: 298.15 K / Instrument: SPOTITON
Details: Samples were frozen with the SPT Labtech chameleon.

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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: 3 / Number real images: 8049 / 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: 0.75 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: 866869
Startup modelType of model: OTHER / Details: Ab Initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 17739
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7ut7


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 49.9
Output model

PDB-9ctz:
Azotobacter vinelandii MoFeP (C2 symmetry)

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