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- PDB-8ew3: Cryo EM structure of Vibrio cholerae NQR -

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Basic information

Entry
Database: PDB / ID: 8ew3
TitleCryo EM structure of Vibrio cholerae NQR
Components(Na(+)-translocating NADH-quinone reductase subunit ...) x 6
KeywordsELECTRON TRANSPORT / TRANSLOCASE / complex
Function / homology
Function and homology information


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 / respiratory electron transport chain / 2 iron, 2 sulfur cluster binding / transmembrane transport / 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
FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN / UBIQUINONE-1 / Na(+)-translocating NADH-quinone reductase subunit E / Na(+)-translocating NADH-quinone reductase subunit C / Na(+)-translocating NADH-quinone reductase subunit B / Na(+)-translocating NADH-quinone reductase subunit F / Na(+)-translocating NADH-quinone reductase subunit A / Na(+)-translocating NADH-quinone reductase subunit D
Similarity search - Component
Biological speciesVibrio cholerae O395 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.65159 Å
AuthorsFuller, J.R. / Juarez, O.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI151152 United States
CitationJournal: To Be Published
Title: Novel cofactor binding motifs, electron transfer and ion pumping mechanisms of the respiratory complex NQR
Authors: Juarez, O. / Fuller, J.R.
History
DepositionOct 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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)212,88711
Polymers211,1726
Non-polymers1,7155
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
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) / Strain: O395N1 / Gene: nqrA, D6U24_04460, EYB64_17955, FLM02_04825 / Production host: Vibrio cholerae O395 (bacteria) / Strain (production host): O395N1
References: UniProt: A0A2D2BC37, 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) / Strain: O395N1
Gene: nqrB, D6U24_04465, ERS013186_02082, ERS013198_02508, ERS013199_02395, ERS013200_04117, ERS013202_01883, ERS013206_02987, ERS013207_01958, EYB64_17950, F0H40_10090, FLM02_04820, FLM12_12920, FXE67_12105
Production host: Vibrio cholerae O395 (bacteria) / Strain (production host): O395N1
References: UniProt: A0A085SSI3, 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) / Strain: O395N1
Gene: nqrC, BC353_01370, D6U24_04470, ERS013165_00616, ERS013186_02083, ERS013198_02507, ERS013199_02396, ERS013200_04118, ERS013201_01110, ERS013202_01884, ERS013206_02988, ERS013207_01959, F0H40_ ...Gene: nqrC, BC353_01370, D6U24_04470, ERS013165_00616, ERS013186_02083, ERS013198_02507, ERS013199_02396, ERS013200_04118, ERS013201_01110, ERS013202_01884, ERS013206_02988, ERS013207_01959, F0H40_10095, FLM02_04815, FLM12_12915
Production host: Vibrio cholerae O395 (bacteria) / Strain (production host): O395N1
References: UniProt: A0A085R7S2, 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) / Strain: O395N1 / Gene: nqrD, VC0395_A1881, VC395_2408 / Production host: Vibrio cholerae O395 (bacteria) / Strain (production host): O395N1
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) / Strain: O395N1
Gene: nqrE, BC353_01360, D6U24_04480, ERS013165_00614, ERS013186_02085, ERS013198_02505, ERS013199_02398, ERS013200_04120, ERS013201_01112, ERS013202_01886, ERS013206_02990, ERS013207_01961, EYB64_ ...Gene: nqrE, BC353_01360, D6U24_04480, ERS013165_00614, ERS013186_02085, ERS013198_02505, ERS013199_02398, ERS013200_04120, ERS013201_01112, ERS013202_01886, ERS013206_02990, ERS013207_01961, EYB64_17935, F0315_08350, F0H40_10105, F0M16_14025, FLM02_04805, FLM12_12905
Production host: Vibrio cholerae O395 (bacteria) / Strain (production host): O395N1
References: UniProt: A0A085QWM0, 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: 45113.492 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae O395 (bacteria) / Strain: O395N1
Gene: nqrF, D6U24_04485, ERS013198_02504, ERS013199_02399, ERS013201_01113, ERS013202_01887, ERS013206_02991, EYB64_17930, FLM12_12900
Production host: Vibrio cholerae O395 (bacteria) / Strain (production host): O395N1
References: UniProt: A0A085ST13, NADH:ubiquinone reductase (Na+-transporting)

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Non-polymers , 4 types, 5 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-UQ1 / UBIQUINONE-1


Mass: 250.290 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H18O4 / Feature type: SUBJECT OF INVESTIGATION
#10: 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

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Details

Has ligand of interestY
Has protein modificationY

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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 / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vibrio cholerae O395 (bacteria) / Strain: O395N1
Source (recombinant)Organism: Vibrio cholerae O395 (bacteria) / Strain: O395N1
Buffer solutionpH: 7 / Details: 100 mM KCl, 1 mM EDTA, 50 mM HEPES, pH 7.0
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
21 mMEDTAC10H16N2O81
350 mMHEPESC8H18N2O4S1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Gatan Solarus plasma cleaner operated at 20W and using ambient/room air
Grid 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: 295 K
Details: A 3.5 uL droplet of sample was applied to the grid surface and blotted for 4 s before plunging into liquid 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 1200 nm / Calibrated defocus max: 3600 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
Image recordingAverage exposure time: 4.66 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3159 / Details: 50 e-/A2 fractionated over 40 movie frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
3EPU2.9image acquisition
5CTFFIND4.1.14CTF correction
6RELION4.0beta2CTF correction
9UCSF Chimeramodel fitting
10RELION4.0beta2initial Euler assignment
11RELION4.0beta2classification
12RELION4.0beta23D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
15Rosettamodel refinement
Image processingDetails: Micrograph movies were summed and dose-weighted using the motion correction algorithm implemented in RELION.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 304335
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.65159 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73763 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation
Details: The model was refined by iterating between manual edits in Coot, molecular dynamics-guided edits using the ISOLDE ChimeraX package, and automated real-space refinement in Phenix. Models that ...Details: The model was refined by iterating between manual edits in Coot, molecular dynamics-guided edits using the ISOLDE ChimeraX package, and automated real-space refinement in Phenix. Models that had been automatically rebuilt using Rosetta were used as guides during manual refinement.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 70.1 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002112170
ELECTRON MICROSCOPYf_angle_d0.413316517
ELECTRON MICROSCOPYf_chiral_restr0.03881899
ELECTRON MICROSCOPYf_plane_restr0.00342071
ELECTRON MICROSCOPYf_dihedral_angle_d8.58424368

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