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- PDB-9udf: Cryo-EM structure of Na+-translocating NADH-ubiquinone oxidoreduc... -

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Basic information

Entry
Database: PDB / ID: 9udf
TitleCryo-EM structure of Na+-translocating NADH-ubiquinone oxidoreductase NqrB-G141A mutant from Vibrio cholerae reduced by NADH, with bound korormicin A, shifted state
Components(Na(+)-translocating NADH-quinone reductase subunit ...) x 6
KeywordsMEMBRANE PROTEIN / Na+-NQR / Na+ pump / oxidoreductase / inhibitor
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 / NqrA second alpha/beta domain ...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 / NqrA second alpha/beta 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
FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / Korormicin / 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 ...FLAVIN-ADENINE DINUCLEOTIDE / FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / Korormicin / 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: 2.93 Å
AuthorsIshikawa-Fukuda, M. / Kishikawa, J. / Kato, T. / Murai, M.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22KJ1795 Japan
Japan Society for the Promotion of Science (JSPS)22H02273 Japan
Japan Society for the Promotion of Science (JSPS)24K08729 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121001 Japan
CitationJournal: bioRxiv / Year: 2025
Title: The Na -pumping mechanism driven by redox reactions in the NADH-quinone oxidoreductase from relies on dynamic conformational changes.
Authors: Moe Ishikawa-Fukuda / Takehito Seki / Jun-Ichi Kishikawa / Masuya Takahiro / Kei-Ichi Okazaki / Takayuki Kato / Blanca Barquera / Hideto Miyoshi / Masatoshi Murai
Abstract: The Na -pumping NADH-quinone oxidoreductase (Na -NQR) is a key respiratory enzyme in many marine and pathogenic bacteria that couples electron transfer to Na -pumping across the membrane. Earlier ...The Na -pumping NADH-quinone oxidoreductase (Na -NQR) is a key respiratory enzyme in many marine and pathogenic bacteria that couples electron transfer to Na -pumping across the membrane. Earlier X-ray and cryo-EM structures of Na -NQR from suggested that the subunits harboring redox cofactors undergo conformational changes during catalytic turnover. However, these proposed rearrangements have not yet been confirmed. Here, we have identified at least five distinct conformational states of Na -NQR using: mutants that lack specific cofactors, specific inhibitors or low-sodium conditions. Molecular dynamics simulations based on these structural insights indicate that 2Fe-2S reduction in NqrD/E plays a crucial role in triggering Na translocation by driving structural rearrangements in the NqrD/E subunits, which subsequently influence NqrC and NqrF positioning. This study provides the first structural insights into the mechanism of Na translocation coupled to electron transfer in Na⁺-NQR.
History
DepositionApr 6, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 25, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)217,42418
Polymers212,0156
Non-polymers5,40912
Water30617
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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: 45404.906 Da / Num. of mol.: 1 / Mutation: G141A
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
Details: C-TERMINAL INCLUDES HIS-TAG SEQUENCE AS A PURIFICATION TAG.
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

#10: 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, 27 molecules

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


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P
#8: Chemical ChemComp-RBF / RIBOFLAVIN / RIBOFLAVINE / VITAMIN B2


Mass: 376.364 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H20N4O6
#9: 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
#11: Chemical ChemComp-IQT / Korormicin


Mass: 433.581 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H39NO5
#12: Chemical ChemComp-CA / CALCIUM ION


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


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


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


Mass: 18.015 Da / Num. of mol.: 17 / 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: Na(+)-translocating NADH-quinone reductase / Type: COMPLEX / Details: NqrB-G141A / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.214 MDa / Experimental value: NO
Source (natural)Organism: Vibrio cholerae O395 (bacteria)
Source (recombinant)Organism: Vibrio cholerae (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClC4H13Cl2NO31
2100 mMSodium chlorideNaCl1
31 mMEthylenediaminetetraacetic acidC10H16N2O81
40.05 %n-DODECYL-beta-D-MALTOSIDEC24H46O111
55 mMNADHC21H27N7O14P21
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: TFS 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: 1800 nm / Nominal defocus min: 800 nm / Cs: 0.001864 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter slit width: 20 eV / Spherical aberration corrector: Image corrector

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.1particle selectionTopaz Extract
2SerialEM4image acquisition
4cryoSPARCv3.3.1CTF correction
7cryoSPARCv3.3.1model fitting
9cryoSPARCv3.3.1initial Euler assignment
10cryoSPARCv3.3.1final Euler assignment
11cryoSPARCv3.3.1classification
12cryoSPARCv3.3.13D reconstruction
19PHENIX1.21_5207model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1384337
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74083 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: The hydrophilic moiety of NqrC is fitted by rigid body fitting. The other is fitted by flexible fitting.
Atomic model buildingPDB-ID: 9LRR

9lrr


Accession code: 9LRR / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.93 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315455
ELECTRON MICROSCOPYf_angle_d0.58420945
ELECTRON MICROSCOPYf_dihedral_angle_d12.6582380
ELECTRON MICROSCOPYf_chiral_restr0.0432356
ELECTRON MICROSCOPYf_plane_restr0.0052619

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