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- PDB-8rbm: Cryo-EM structure of the NADH:ferredoxin oxidoreductase RNF from ... -

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Basic information

Entry
Database: PDB / ID: 8rbm
TitleCryo-EM structure of the NADH:ferredoxin oxidoreductase RNF from Azotobacter vinelandii, ferricyanide oxidized
Components(Ion-translocating oxidoreductase complex subunit ...) x 6
KeywordsMEMBRANE PROTEIN / NADH:ferredoxin oxidoreductase
Function / homology
Function and homology information


Translocases / electron transport chain / transmembrane transport / FMN binding / 4 iron, 4 sulfur cluster binding / electron transfer activity / metal ion binding / plasma membrane
Similarity search - Function
Ion-translocating oxidoreductase complex, RnfB/RsxB / Ion-translocating oxidoreductase complex, subunit RnfC/RsxC / Ion-translocating oxidoreductase complex subunit E / Ion-translocating oxidoreductase complex, subunit RnfA/RsxA / Ion-translocating oxidoreductase complex Rnf, subunit D / Electron transport complex, RnfB/RsxB, Proteobacteria / RnfC Barrel sandwich hybrid domain / RnfC Barrel sandwich hybrid domain / Ion-translocating oxidoreductase complex, subunit RnfG/RsxG / 4Fe-4S domain ...Ion-translocating oxidoreductase complex, RnfB/RsxB / Ion-translocating oxidoreductase complex, subunit RnfC/RsxC / Ion-translocating oxidoreductase complex subunit E / Ion-translocating oxidoreductase complex, subunit RnfA/RsxA / Ion-translocating oxidoreductase complex Rnf, subunit D / Electron transport complex, RnfB/RsxB, Proteobacteria / RnfC Barrel sandwich hybrid domain / RnfC Barrel sandwich hybrid domain / Ion-translocating oxidoreductase complex, subunit RnfG/RsxG / 4Fe-4S domain / Putative Fe-S cluster / 4Fe-4S domain profile. / 4Fe-4S dicluster domain / NqrDE/RnfAE / Ion-translocating oxidoreductase NqrB/RnfD / Rnf-Nqr subunit, membrane protein / NQR2, RnfD, RnfE family / FMN-binding / FMN-binding domain / FMN_bind / 4Fe-4S dicluster domain / Soluble ligand binding domain / SLBB domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Respiratory-chain NADH dehydrogenase 51 Kd subunit / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain
Similarity search - Domain/homology
FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / PHOSPHATIDYLETHANOLAMINE / RIBOFLAVIN / IRON/SULFUR CLUSTER / Ion-translocating oxidoreductase complex subunit G / Ion-translocating oxidoreductase complex subunit D / Ion-translocating oxidoreductase complex subunit C / Ion-translocating oxidoreductase complex subunit B / Ion-translocating oxidoreductase complex subunit A / Ion-translocating oxidoreductase complex subunit E
Similarity search - Component
Biological speciesAzotobacter vinelandii DJ (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsZhang, L. / Einsle, O.
Funding supportEuropean Union, Germany, 5items
OrganizationGrant numberCountry
European Research Council (ERC)310656European Union
German Research Foundation (DFG)CRC 1381, project ID 403222702 Germany
German Research Foundation (DFG)CRC 992, project ID 192904750 Germany
German Research Foundation (DFG)RTG 2202, project ID 46710898 Germany
German Research Foundation (DFG)grant INST 35/134-1 FUGG Germany
CitationJournal: Nat Chem Biol / Year: 2024
Title: Architecture of the RNF1 complex that drives biological nitrogen fixation.
Authors: Lin Zhang / Oliver Einsle /
Abstract: Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living ...Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryogenic electron microscopy structure of the nitrogenase-associated RNF complex of A. vinelandii, a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron-sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling.
History
DepositionDec 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ion-translocating oxidoreductase complex subunit A
C: Ion-translocating oxidoreductase complex subunit C
D: Ion-translocating oxidoreductase complex subunit D
E: Ion-translocating oxidoreductase complex subunit E
G: Ion-translocating oxidoreductase complex subunit G
B: Ion-translocating oxidoreductase complex subunit B
b: Ion-translocating oxidoreductase complex subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)206,72727
Polymers197,2417
Non-polymers9,48620
Water00
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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Ion-translocating oxidoreductase complex subunit ... , 6 types, 7 molecules ACDEGBb

#1: Protein Ion-translocating oxidoreductase complex subunit A / Rnf electron transport complex subunit A


Mass: 19942.131 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: rnfA1, rnfA, Avin_50980 / Production host: Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DMA8, Translocases
#2: Protein Ion-translocating oxidoreductase complex subunit C / Rnf electron transport complex subunit C


Mass: 52234.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: rnfC1, rnfC, Avin_50960 / Production host: Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DMA6, Translocases
#3: Protein Ion-translocating oxidoreductase complex subunit D / Rnf electron transport complex subunit D


Mass: 39189.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: rnfD1, rnfD, Avin_50950 / Production host: Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DMA5, Translocases
#4: Protein Ion-translocating oxidoreductase complex subunit E / Rnf electron transport complex subunit E


Mass: 25576.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: rnfE, Avin_50930 / Production host: Azotobacter vinelandii DJ (bacteria) / References: UniProt: Q9F5Y1, Translocases
#5: Protein Ion-translocating oxidoreductase complex subunit G / Rnf electron transport complex subunit G


Mass: 24890.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: rnfG1, rnfG, Avin_50940 / Production host: Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DMA4, Translocases
#6: Protein Ion-translocating oxidoreductase complex subunit B / Rnf electron transport complex subunit B


Mass: 17704.572 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Gene: rnfB1, rnfB, Avin_50970 / Production host: Azotobacter vinelandii DJ (bacteria) / References: UniProt: C1DMA7, Translocases

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Sugars , 1 types, 12 molecules

#8: Sugar
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C24H46O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM

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Non-polymers , 5 types, 8 molecules

#7: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C17H21N4O9P / Source: (gene. exp.) Azotobacter vinelandii DJ (bacteria) / Production host: Azotobacter vinelandii DJ (bacteria) / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-RBF / RIBOFLAVIN / RIBOFLAVINE / VITAMIN B2


Mass: 376.364 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H20N4O6 / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM

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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: Heptameric complex NADH:ferredoxin oxidoreductase RNF1
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.188 MDa / Experimental value: NO
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Source (recombinant)Organism: Azotobacter vinelandii DJ (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 37 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166974 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311515
ELECTRON MICROSCOPYf_angle_d0.55215685
ELECTRON MICROSCOPYf_dihedral_angle_d11.4634116
ELECTRON MICROSCOPYf_chiral_restr0.041909
ELECTRON MICROSCOPYf_plane_restr0.0051903

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