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- PDB-8crs: CryoEM Structure of nitrogenase MoFe-protein in detergent -

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Basic information

Entry
Database: PDB / ID: 8crs
TitleCryoEM Structure of nitrogenase MoFe-protein in detergent
Components(Nitrogenase molybdenum-iron protein ...) x 2
KeywordsMETAL BINDING PROTEIN / OXIDOREDUCTASE / Nitrogenase / metalloenzyme
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: 2.04 Å
AuthorsWarmack, R.A. / Rees, D.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM045162 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143836-01 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural consequences of turnover-induced homocitrate loss in nitrogenase.
Authors: Rebeccah A Warmack / Ailiena O Maggiolo / Andres Orta / Belinda B Wenke / James B Howard / Douglas C Rees /
Abstract: Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor ...Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.
History
DepositionMay 11, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 8, 2023Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.2May 21, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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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)233,35816
Polymers227,3894
Non-polymers5,96912
Water30,4811692
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 54289.906 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: 59404.684 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 , 6 types, 1704 molecules

#3: Chemical ChemComp-ICS / iron-sulfur-molybdenum cluster with interstitial carbon


Mass: 787.451 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: CFe7MoS9
#4: Chemical ChemComp-HCA / 3-HYDROXY-3-CARBOXY-ADIPIC ACID


Mass: 206.150 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C7H10O7
#5: Chemical
ChemComp-1N7 / CHAPSO / 2-hydroxy-N,N-dimethyl-3-sulfo-N-(3-{[(3beta,5beta,7beta,12beta)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)propan-1-aminium


Mass: 631.884 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C32H59N2O8S / Comment: detergent*YM
#6: Chemical ChemComp-CLF / FE(8)-S(7) CLUSTER


Mass: 671.215 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe8S7
#7: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1692 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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 nitrogenase MoFe-protein / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Source (natural)Organism: Azotobacter vinelandii (bacteria)
Buffer solutionpH: 7.8
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: -3000 nm / Nominal defocus min: -800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137629 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416610
ELECTRON MICROSCOPYf_angle_d0.61322628
ELECTRON MICROSCOPYf_dihedral_angle_d6.2882326
ELECTRON MICROSCOPYf_chiral_restr0.0472398
ELECTRON MICROSCOPYf_plane_restr0.0052864

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