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- PDB-7ut7: C2 symmetric cryoEM structure of Azotobacter vinelandii MoFeP und... -

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Entry
Database: PDB / ID: 7ut7
TitleC2 symmetric cryoEM structure of Azotobacter vinelandii MoFeP under non-turnover conditions
Components(Nitrogenase molybdenum-iron protein ...) x 2
KeywordsOXIDOREDUCTASE / nitrogenase / MoFeP / nitrogen fixation
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 beta chain / Nitrogenase molybdenum-iron protein alpha chain
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.91 Å
AuthorsRutledge, H.L. / Cook, B.D. / Tezcan, F.A. / Herzik, M.A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM099813 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138206 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM008326 United States
National Aeronautic Space Administration (NASA, United States)80NSSC18M0093 United States
CitationJournal: Science / Year: 2022
Title: Structures of the nitrogenase complex prepared under catalytic turnover conditions.
Authors: Hannah L Rutledge / Brian D Cook / Hoang P M Nguyen / Mark A Herzik / F Akif Tezcan /
Abstract: The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ...The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.
History
DepositionApr 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 31, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
Water7,476415
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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: 55363.043 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii DJ (bacteria) / References: UniProt: P07328, nitrogenase
#2: Protein Nitrogenase molybdenum-iron protein beta chain / Dinitrogenase


Mass: 59535.879 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DGZ8, nitrogenase

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Non-polymers , 5 types, 423 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
#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.: 415 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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 MoFeP from Azotobacter vinelandii / Type: COMPLEX
Details: Wild-type MoFeP was purified from the native organism, Azotobacter vinelandii
Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.23321 MDa / Experimental value: NO
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Buffer solutionpH: 8
Details: Solutions were prepared and filtered immediately prior to the experiment.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRIS(HOCH2)3CNH21
225 mMsodium chlorideNaCl1
SpecimenConc.: 1.44 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K / Details: Custom manual plunger. Greater than 95% humidity.

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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: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 123 K / Temperature (min): 93 K
Image recordingElectron dose: 65 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3773

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2RELIONparticle selection
3EPU2image acquisition
5CTFFIND4.1CTF correction
11RELION4.0/beta2initial Euler assignment
12RELION4.0/beta2final Euler assignment
13cryoSPARC3.3.2classification
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2628629
3D reconstructionResolution: 1.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177123 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416500
ELECTRON MICROSCOPYf_angle_d0.63522773
ELECTRON MICROSCOPYf_dihedral_angle_d5.2392291
ELECTRON MICROSCOPYf_chiral_restr0.0472364
ELECTRON MICROSCOPYf_plane_restr0.0052859

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