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- PDB-6wti: The Cryo-EM structure of the ubiquinol oxidase from Escherichia coli -

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Basic information

Entry
Database: PDB / ID: 6wti
TitleThe Cryo-EM structure of the ubiquinol oxidase from Escherichia coli
Components
  • (Cytochrome o ubiquinol oxidase, subunit ...) x 2
  • Cytochrome o ubiquinol oxidase
  • Ubiquinol oxidase subunit 2
KeywordsOXIDOREDUCTASE / ubiquinol oxidase cytochrome bo3 / membrane protein
Function / homology
Function and homology information


cytochrome bo3 ubiquinol oxidase activity => GO:0009486 / cytochrome o ubiquinol oxidase complex / oxidoreduction-driven active transmembrane transporter activity / ubiquinol oxidase (H+-transporting) / cytochrome bo3 ubiquinol oxidase activity / aerobic electron transport chain / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / ubiquinone binding / electron transport coupled proton transport / cytochrome-c oxidase activity ...cytochrome bo3 ubiquinol oxidase activity => GO:0009486 / cytochrome o ubiquinol oxidase complex / oxidoreduction-driven active transmembrane transporter activity / ubiquinol oxidase (H+-transporting) / cytochrome bo3 ubiquinol oxidase activity / aerobic electron transport chain / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / ubiquinone binding / electron transport coupled proton transport / cytochrome-c oxidase activity / proton transmembrane transporter activity / ATP synthesis coupled electron transport / respirasome / aerobic respiration / respiratory electron transport chain / electron transfer activity / membrane => GO:0016020 / copper ion binding / heme binding / plasma membrane
Similarity search - Function
Cytochrome o ubiquinol oxidase, subunit III / Cytochrome o ubiquinol oxidase subunit IV / Cytochrome o ubiquinol oxidase, subunit I / Ubiquinol oxidase subunit III domain / Cytochrome C oxidase subunit IV, prokaryotes / COX aromatic rich motif / Prokaryotic Cytochrome C oxidase subunit IV / COX Aromatic Rich Motif / Cytochrome o ubiquinol oxidase subunit II / Ubiquinol oxidase subunit 2, cupredoxin domain ...Cytochrome o ubiquinol oxidase, subunit III / Cytochrome o ubiquinol oxidase subunit IV / Cytochrome o ubiquinol oxidase, subunit I / Ubiquinol oxidase subunit III domain / Cytochrome C oxidase subunit IV, prokaryotes / COX aromatic rich motif / Prokaryotic Cytochrome C oxidase subunit IV / COX Aromatic Rich Motif / Cytochrome o ubiquinol oxidase subunit II / Ubiquinol oxidase subunit 2, cupredoxin domain / Cytochrome c oxidase subunit III / Cytochrome c oxidase subunit III-like / Cytochrome c oxidase, subunit III, 4-helical bundle / Cytochrome c oxidase subunit III / Heme-copper oxidase subunit III family profile. / Cytochrome c oxidase subunit III-like superfamily / Cytochrome C oxidase subunit II, transmembrane domain / Cytochrome oxidase subunit II transmembrane region profile. / Cytochrome c/quinol oxidase subunit II / Cytochrome C oxidase subunit II, transmembrane domain superfamily / Cytochrome c oxidase, subunit I, copper-binding site / Heme-copper oxidase catalytic subunit, copper B binding region signature. / Cytochrome c oxidase-like, subunit I domain / Cytochrome oxidase subunit I profile. / Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I / Cytochrome C oxidase subunit II, periplasmic domain / Cytochrome c oxidase subunit II-like C-terminal / Cytochrome oxidase subunit II copper A binding domain profile. / Cupredoxin / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
1,2-Distearoyl-sn-glycerophosphoethanolamine / COPPER (II) ION / PROTOPORPHYRIN IX CONTAINING FE / HEME O / pentadecyl(tetradecyl)peroxyanhydride / Ubiquinone-8 / Ubiquinol oxidase subunit 2 / Cytochrome bo(3) ubiquinol oxidase subunit 3 / Cytochrome bo(3) ubiquinol oxidase subunit 1 / Cytochrome bo(3) ubiquinol oxidase subunit 4 ...1,2-Distearoyl-sn-glycerophosphoethanolamine / COPPER (II) ION / PROTOPORPHYRIN IX CONTAINING FE / HEME O / pentadecyl(tetradecyl)peroxyanhydride / Ubiquinone-8 / Ubiquinol oxidase subunit 2 / Cytochrome bo(3) ubiquinol oxidase subunit 3 / Cytochrome bo(3) ubiquinol oxidase subunit 1 / Cytochrome bo(3) ubiquinol oxidase subunit 4 / Cytochrome bo(3) ubiquinol oxidase subunit 1 / Cytochrome bo(3) ubiquinol oxidase subunit 2 / Cytochrome bo(3) ubiquinol oxidase subunit 3 / Cytochrome bo(3) ubiquinol oxidase subunit 4
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.38 Å
AuthorsSu, C.-C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Nat Methods / Year: 2021
Title: A 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins.
Authors: Chih-Chia Su / Meinan Lyu / Christopher E Morgan / Jani Reddy Bolla / Carol V Robinson / Edward W Yu /
Abstract: Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein ...Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.
History
DepositionMay 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 20, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.pdbx_description / _pdbx_entity_nonpoly.name

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Structure visualization

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Assembly

Deposited unit
A: Cytochrome o ubiquinol oxidase, subunit I
B: Ubiquinol oxidase subunit 2
C: Cytochrome o ubiquinol oxidase
D: Cytochrome o ubiquinol oxidase, subunit IV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,55518
Polymers144,0524
Non-polymers9,50314
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28250 Å2
ΔGint-336 kcal/mol
Surface area42100 Å2

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Components

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Cytochrome o ubiquinol oxidase, subunit ... , 2 types, 2 molecules AD

#1: Protein Cytochrome o ubiquinol oxidase, subunit I


Mass: 74424.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cyoB, ECDEC2C_0430 / Production host: Escherichia coli (E. coli)
References: UniProt: H4KCU1, UniProt: P0ABI8*PLUS, Oxidoreductases; Acting on diphenols and related substances as donors; With oxygen as acceptor
#4: Protein Cytochrome o ubiquinol oxidase, subunit IV


Mass: 12037.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cyoD, EC12741_3890 / Production host: Escherichia coli (E. coli)
References: UniProt: I2RK84, UniProt: P0ABJ6*PLUS, Oxidoreductases; Acting on diphenols and related substances as donors; With oxygen as acceptor

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Protein , 2 types, 2 molecules BC

#2: Protein Ubiquinol oxidase subunit 2 /


Mass: 34947.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cyoA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A024L5V9, UniProt: P0ABJ1*PLUS
#3: Protein Cytochrome o ubiquinol oxidase


Mass: 22642.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ECDG_00226 / Production host: Escherichia coli (E. coli) / References: UniProt: D6I7E4, UniProt: P0ABJ3*PLUS

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Non-polymers , 6 types, 14 molecules

#5: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#6: Chemical ChemComp-HEO / HEME O / Heme O


Mass: 838.854 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C49H58FeN4O5
#7: Chemical ChemComp-CU / COPPER (II) ION / Copper


Mass: 63.546 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-UQ8 / Ubiquinone-8 / 2,3-dimethoxy-5-methyl-6-[(6E,10E,14E,18E,22E,26E)-3,7,11,15,19,23,27,31-octamethyldotriaconta-2,6,10,14,18,22,26,30-oc taen-1-yl]cyclohexa-2,5-diene-1,4-dione


Mass: 727.109 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C49H74O4
#9: Chemical
ChemComp-3PE / 1,2-Distearoyl-sn-glycerophosphoethanolamine / 3-SN-PHOSPHATIDYLETHANOLAMINE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / Phosphatidylethanolamine


Mass: 748.065 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C41H82NO8P / Comment: phospholipid*YM
#10: Chemical ChemComp-U9V / pentadecyl(tetradecyl)peroxyanhydride


Mass: 524.859 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H64O4

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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: ubiquinol oxidase / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 334222 / Symmetry type: POINT

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