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- PDB-8dpn: CryoEM structure of Azotobacter vinelandii nitrogenase MoFeP duri... -

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Basic information

Entry
Database: PDB / ID: 8dpn
TitleCryoEM structure of Azotobacter vinelandii nitrogenase MoFeP during catalytic N2 reduction
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: 2.49 Å
AuthorsRutledge, H.L. / Cook, B. / 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
DepositionJul 15, 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 / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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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
Water72140
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: The MoFeP tetramer is part of a heterogeneous mixture of complexes on the grid.
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, 48 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-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
#6: Chemical ChemComp-FE / FE (III) ION


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


Mass: 18.015 Da / Num. of mol.: 40 / 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: Azotobacter vinelandii nitrogenase MoFeP during catalytic N2 reduction
Type: COMPLEX
Details: Wild-type MoFeP was purified from the native organism, Azotobacter vinelandii. This map is the structure of free MoFeP from during catalytic N2 reduction.
Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.29621 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.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 1.4 mg/mL MoFeP 3.6 mg/mL FeP Note that this structure does not contain FeP
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: 135000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 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 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14903

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPU2.8image acquisition
4CTFFIND4.1CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11RELION4.0/beta2initial Euler assignment
12RELION4.0/beta2final Euler assignment
13cryoSPARC3.32classification
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 19711170
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29181 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 4WZA

4wza


Accession code: 4WZA / Source name: PDB / Type: experimental model

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