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- PDB-7xk6: Cryo-EM structure of Na+-pumping NADH-ubiquinone oxidoreductase f... -

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Basic information

Entry
Database: PDB / ID: 7xk6
TitleCryo-EM structure of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae, with aurachin D-42
Components(Na(+)-translocating NADH-quinone reductase subunit ...) x 6
KeywordsTRANSLOCASE / NADH-quinone oxidoreductase / redox-driven sodium pump / bioenergetics / Vibrio cholerae / electron transport / membrane protein complex / OXIDOREDUCTASE
Function / homology
Function and homology information


riboflavin binding / NADH:ubiquinone reductase (Na+-transporting) / Gram-negative-bacterium-type cell wall / oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor / sodium ion transport / FAD binding / respiratory electron transport chain / transmembrane transport / 2 iron, 2 sulfur cluster binding / FMN binding ...riboflavin binding / NADH:ubiquinone reductase (Na+-transporting) / Gram-negative-bacterium-type cell wall / oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor / sodium ion transport / FAD binding / respiratory electron transport chain / transmembrane transport / 2 iron, 2 sulfur cluster binding / FMN binding / electron transfer activity / metal ion binding / plasma membrane
Similarity search - Function
Na(+)-translocating NADH-quinone reductase subunit B / Na(+)-translocating NADH-quinone reductase subunit E / Na(+)-translocating NADH-quinone reductase subunit D / Na(+)-translocating NADH-quinone reductase subunit A / Na(+)-translocating NADH-quinone reductase subunit A, C-terminal domain / NqrA N-terminal barrel-sandwich hybrid domain / NQRA C-terminal domain / Na(+)-translocating NADH-quinone reductase subunit C / Na(+)-translocating NADH-quinone reductase subunit F / NqrDE/RnfAE ...Na(+)-translocating NADH-quinone reductase subunit B / Na(+)-translocating NADH-quinone reductase subunit E / Na(+)-translocating NADH-quinone reductase subunit D / Na(+)-translocating NADH-quinone reductase subunit A / Na(+)-translocating NADH-quinone reductase subunit A, C-terminal domain / NqrA N-terminal barrel-sandwich hybrid domain / NQRA C-terminal domain / Na(+)-translocating NADH-quinone reductase subunit C / Na(+)-translocating NADH-quinone reductase subunit F / NqrDE/RnfAE / Ion-translocating oxidoreductase NqrB/RnfD / : / Rnf-Nqr subunit, membrane protein / NQR2, RnfD, RnfE family / FMN-binding / FMN-binding domain / FMN_bind / Flavoprotein pyridine nucleotide cytochrome reductase-like, FAD-binding domain / Oxidoreductase FAD-binding domain / 2Fe-2S iron-sulfur cluster binding domain / Beta-grasp domain superfamily / 2Fe-2S ferredoxin-type iron-sulfur binding domain profile. / 2Fe-2S ferredoxin-type iron-sulfur binding domain / 2Fe-2S ferredoxin-like superfamily / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel
Similarity search - Domain/homology
Aurachin D / FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / RIBOFLAVIN / Na(+)-translocating NADH-quinone reductase subunit B / Na(+)-translocating NADH-quinone reductase subunit A / Na(+)-translocating NADH-quinone reductase subunit F / Na(+)-translocating NADH-quinone reductase subunit E ...Aurachin D / FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / RIBOFLAVIN / Na(+)-translocating NADH-quinone reductase subunit B / Na(+)-translocating NADH-quinone reductase subunit A / Na(+)-translocating NADH-quinone reductase subunit F / Na(+)-translocating NADH-quinone reductase subunit E / Na(+)-translocating NADH-quinone reductase subunit D / Na(+)-translocating NADH-quinone reductase subunit C
Similarity search - Component
Biological speciesVibrio cholerae O395 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsKishikawa, J. / Ishikawa, M. / Masuya, T. / Murai, M. / Barquera, B. / Miyoshi, H.
Funding support Japan, United States, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20K06514 Japan
Japan Society for the Promotion of Science (JSPS)21H02130 Japan
Japan Society for the Promotion of Science (JSPS)22K14837 Japan
Japan Society for the Promotion of Science (JSPS)22H02273 Japan
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01-AI132580 United States
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of Na-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.
Authors: Jun-Ichi Kishikawa / Moe Ishikawa / Takahiro Masuya / Masatoshi Murai / Yuki Kitazumi / Nicole L Butler / Takayuki Kato / Blanca Barquera / Hideto Miyoshi /
Abstract: The Na-pumping NADH-ubiquinone oxidoreductase (Na-NQR) couples electron transfer from NADH to ubiquinone with Na-pumping, generating an electrochemical Na gradient that is essential for energy- ...The Na-pumping NADH-ubiquinone oxidoreductase (Na-NQR) couples electron transfer from NADH to ubiquinone with Na-pumping, generating an electrochemical Na gradient that is essential for energy-consuming reactions in bacteria. Since Na-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state. Here we present cryo-electron microscopy structures of Na-NQR from Vibrio cholerae with or without a bound inhibitor at 2.5- to 3.1-Å resolution. The structures reveal the arrangement of all six redox cofactors including a herein identified 2Fe-2S cluster located between the NqrD and NqrE subunits. A large part of the hydrophilic NqrF is barely visible in the density map, suggesting a high degree of flexibility. This flexibility may be responsible to reducing the long distance between the 2Fe-2S centers in NqrF and NqrD/E. Two different types of specific inhibitors bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The present study provides a foundation for understanding the function of Na-NQR and the binding manner of specific inhibitors.
History
DepositionApr 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Na(+)-translocating NADH-quinone reductase subunit A
B: Na(+)-translocating NADH-quinone reductase subunit B
C: Na(+)-translocating NADH-quinone reductase subunit C
D: Na(+)-translocating NADH-quinone reductase subunit D
E: Na(+)-translocating NADH-quinone reductase subunit E
F: Na(+)-translocating NADH-quinone reductase subunit F
hetero molecules


Theoretical massNumber of molelcules
Total (without water)217,34018
Polymers212,0016
Non-polymers5,33912
Water88349
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Na(+)-translocating NADH-quinone reductase subunit ... , 6 types, 6 molecules ABCDEF

#1: Protein Na(+)-translocating NADH-quinone reductase subunit A / Na(+)-NQR subunit A / Na(+)-translocating NQR subunit A / NQR complex subunit A / NQR-1 subunit A


Mass: 48680.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Gene: nqrA, VC0395_A1884, VC395_2411 / Production host: Vibrio cholerae (bacteria)
References: UniProt: A5F5X1, NADH:ubiquinone reductase (Na+-transporting)
#2: Protein Na(+)-translocating NADH-quinone reductase subunit B / Na(+)-NQR subunit B / Na(+)-translocating NQR subunit B / NQR complex subunit B / NQR-1 subunit B


Mass: 45390.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Gene: nqrB, VC0395_A1883, VC395_2410 / Production host: Vibrio cholerae (bacteria)
References: UniProt: A5F5X0, NADH:ubiquinone reductase (Na+-transporting)
#3: Protein Na(+)-translocating NADH-quinone reductase subunit C / Na(+)-NQR subunit C / Na(+)-translocating NQR subunit C / NQR complex subunit C / NQR-1 subunit C


Mass: 27652.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Gene: nqrC, VC0395_A1882, VC395_2409 / Production host: Vibrio cholerae (bacteria)
References: UniProt: A5F5Y7, NADH:ubiquinone reductase (Na+-transporting)
#4: Protein Na(+)-translocating NADH-quinone reductase subunit D / Na(+)-NQR subunit D / Na(+)-translocating NQR subunit D / NQR complex subunit D / NQR-1 subunit D


Mass: 22853.217 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Gene: nqrD, VC0395_A1881, VC395_2408 / Production host: Vibrio cholerae (bacteria)
References: UniProt: A5F5Y6, NADH:ubiquinone reductase (Na+-transporting)
#5: Protein Na(+)-translocating NADH-quinone reductase subunit E / Na(+)-NQR subunit E / Na(+)-translocating NQR subunit E / NQR complex subunit E / NQR-1 subunit E


Mass: 21481.678 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Gene: nqrE, VC0395_A1880, VC395_2407 / Production host: Vibrio cholerae (bacteria)
References: UniProt: A5F5Y5, NADH:ubiquinone reductase (Na+-transporting)
#6: Protein Na(+)-translocating NADH-quinone reductase subunit F / Na(+)-NQR subunit F / Na(+)-translocating NQR subunit F / NQR complex subunit F / NQR-1 subunit F


Mass: 45942.363 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Gene: nqrF, VC0395_A1879, VC395_2406 / Production host: Vibrio cholerae (bacteria)
References: UniProt: A5F5Y4, NADH:ubiquinone reductase (Na+-transporting)

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Sugars , 1 types, 2 molecules

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


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 8 types, 59 molecules

#7: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#8: 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
#9: Chemical ChemComp-0NI / Aurachin D / 2-methyl-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-1H-quinolin-4-one


Mass: 363.536 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H33NO / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#12: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#13: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#14: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#15: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 49 / 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: Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae, with aurachin D-42
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: YES
Source (natural)Organism: Vibrio cholerae O395 (bacteria)
Source (recombinant)Organism: Vibrio cholerae (bacteria) / Plasmid: pBAD
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HCl1
2100 mMNaCl1
31 mMEDTA1
40.05 %DDM1
SpecimenConc.: 11 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 0.092 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.5 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11430
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4cryoSPARC3.3.1CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 299013
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54284 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 4P6V

4p6v
PDB Unreleased entry

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415420
ELECTRON MICROSCOPYf_angle_d0.52920896
ELECTRON MICROSCOPYf_dihedral_angle_d6.2769805
ELECTRON MICROSCOPYf_chiral_restr0.0422351
ELECTRON MICROSCOPYf_plane_restr0.0052607

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