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

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Basic information

Entry
Database: PDB / ID: 9ctz
TitleAzotobacter vinelandii MoFeP (C2 symmetry)
Components(Nitrogenase molybdenum-iron protein ...) x 2
KeywordsMETAL BINDING PROTEIN / Nitrogenase / FeMoCo / nitrogen / P-cluster
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
FE(8)-S(7) CLUSTER / : / 3-HYDROXY-3-CARBOXY-ADIPIC ACID / Chem-ICS / Nitrogenase molybdenum-iron protein alpha chain / Nitrogenase molybdenum-iron protein beta chain
Similarity search - Component
Biological speciesAzotobacter vinelandii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsNarehood, S.M. / Cook, B.D. / Srisantitham, S. / Eng, V.H. / Shiau, A. / Britt, R.D. / Herzik, M.A. / Tezcan, F.A.
Funding support United States, 3items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Feb 5, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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 molybdenum-iron protein alpha chain
B: Nitrogenase molybdenum-iron protein beta chain
C: Nitrogenase molybdenum-iron protein alpha chain
D: Nitrogenase molybdenum-iron protein beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,23912
Polymers229,7984
Non-polymers3,4418
Water16,646924
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

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

#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 (bacteria) / References: UniProt: P07328, nitrogenase
#2: Protein Nitrogenase molybdenum-iron protein beta chain / Dinitrogenase / Nitrogenase component I


Mass: 59535.879 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii (bacteria) / References: UniProt: P07329, nitrogenase

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Non-polymers , 5 types, 932 molecules

#3: 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
#4: 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
#5: 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
#6: 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
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 924 / 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: Azotobacter vinelandii MoFeP (C2 symmetry) / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.230 MDa / Experimental value: NO
Source (natural)Organism: Azotobacter vinelandii (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
225 mMSodium ChlorideNaCl1
35 mMSodium DithioniteNa2S2O41
45 mMMagnesium chlorideMgCl21
55 mMAdenosine triphosphateC10H16N5O13P31
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 298.15 K / Details: Samples were 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: 750 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: 3 / Num. of real images: 8049
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
7PHENIXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 866869
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17739 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 49.9 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7UT7

7ut7


Accession code: 7UT7 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316449
ELECTRON MICROSCOPYf_angle_d0.4922315
ELECTRON MICROSCOPYf_dihedral_angle_d5.0082204
ELECTRON MICROSCOPYf_chiral_restr0.0432336
ELECTRON MICROSCOPYf_plane_restr0.0042858

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