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- PDB-8qqk: Cryo-EM structure of E. coli cytochrome bo3 quinol oxidase assemb... -

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Basic information

Entry
Database: PDB / ID: 8qqk
TitleCryo-EM structure of E. coli cytochrome bo3 quinol oxidase assembled in peptidiscs
Components(Cytochrome bo(3) ubiquinol oxidase subunit ...) x 4
KeywordsMEMBRANE PROTEIN / E. coli / Ni-NTA resin / cytochrome bo3 quinol oxidase / ubiquinone-8 release / peptidisc / single particle analysis / cryo-EM
Function / homology
Function and homology information


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 / cytochrome-c oxidase activity / ubiquinone binding / electron transport coupled proton transport / proton transmembrane transporter activity ...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 / cytochrome-c oxidase activity / ubiquinone binding / electron transport coupled proton transport / proton transmembrane transporter activity / ATP synthesis coupled electron transport / respirasome / aerobic respiration / respiratory electron transport chain / electron transfer activity / 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 / Chem-POV / 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 BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsGao, Y. / Zhang, Y. / Hakke, S. / Peters, P.J. / Ravelli, R.B.G.
Funding support Netherlands, 3items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)731.016.407 Netherlands
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
Health-HollandLSHM21029 Netherlands
CitationJournal: Biochim Biophys Acta Bioenerg / Year: 2024
Title: Cryo-EM structure of cytochrome bo quinol oxidase assembled in peptidiscs reveals an "open" conformation for potential ubiquinone-8 release.
Authors: Ye Gao / Yue Zhang / Sneha Hakke / Ronny Mohren / Lyanne J P M Sijbers / Peter J Peters / Raimond B G Ravelli /
Abstract: Cytochrome bo quinol oxidase belongs to the heme‑copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt ...Cytochrome bo quinol oxidase belongs to the heme‑copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1-41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a "U-shape" assembly in contrast to the transmembrane helix, named "TM0", in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo conformations should exist. Our structural model presents experimental evidence for an "open" conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science.
History
DepositionOct 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytochrome bo(3) ubiquinol oxidase subunit 1
B: Cytochrome bo(3) ubiquinol oxidase subunit 2
C: Cytochrome bo(3) ubiquinol oxidase subunit 3
D: Cytochrome bo(3) ubiquinol oxidase subunit 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,09513
Polymers144,0524
Non-polymers6,0439
Water0
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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Cytochrome bo(3) ubiquinol oxidase subunit ... , 4 types, 4 molecules ABCD

#1: Protein Cytochrome bo(3) ubiquinol oxidase subunit 1 / cyt bo(3) subunit 1 / Cytochrome b562-o complex subunit I / Cytochrome o ubiquinol oxidase subunit ...cyt bo(3) subunit 1 / Cytochrome b562-o complex subunit I / Cytochrome o ubiquinol oxidase subunit 1 / Cytochrome o subunit 1 / Oxidase bo(3) subunit 1 / Ubiquinol oxidase chain A / Ubiquinol oxidase polypeptide I / Ubiquinol oxidase subunit 1


Mass: 74424.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Variant: C41
References: UniProt: P0ABI8, ubiquinol oxidase (H+-transporting)
#2: Protein Cytochrome bo(3) ubiquinol oxidase subunit 2 / Cytochrome b562-o complex subunit II / Cytochrome o ubiquinol oxidase subunit 2 / Cytochrome o ...Cytochrome b562-o complex subunit II / Cytochrome o ubiquinol oxidase subunit 2 / Cytochrome o subunit 2 / Oxidase bo(3) subunit 2 / Ubiquinol oxidase chain B / Ubiquinol oxidase polypeptide II / Ubiquinol oxidase subunit 2


Mass: 34947.203 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Variant: C41 / References: UniProt: P0ABJ1
#3: Protein Cytochrome bo(3) ubiquinol oxidase subunit 3 / Cytochrome o ubiquinol oxidase subunit 3 / Cytochrome o subunit 3 / Oxidase bo(3) subunit 3 / ...Cytochrome o ubiquinol oxidase subunit 3 / Cytochrome o subunit 3 / Oxidase bo(3) subunit 3 / Ubiquinol oxidase chain C / Ubiquinol oxidase polypeptide III / Ubiquinol oxidase subunit 3


Mass: 22642.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Variant: C41 / References: UniProt: P0ABJ3
#4: Protein Cytochrome bo(3) ubiquinol oxidase subunit 4 / Cytochrome o ubiquinol oxidase subunit 4 / Cytochrome o subunit 4 / Oxidase bo(3) subunit 4 / ...Cytochrome o ubiquinol oxidase subunit 4 / Cytochrome o subunit 4 / Oxidase bo(3) subunit 4 / Ubiquinol oxidase chain D / Ubiquinol oxidase polypeptide IV / Ubiquinol oxidase subunit 4


Mass: 12037.402 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Variant: C41 / References: UniProt: P0ABJ6

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Non-polymers , 5 types, 9 molecules

#5: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#6: 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.: 3 / Source method: obtained synthetically / Formula: C41H82NO8P / Comment: phospholipid*YM
#7: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


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


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


Mass: 63.546 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cu

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Details

Has ligand of interestN

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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: Cytochrome bo3 quinol oxidase / Type: COMPLEX
Details: Heme-copper-oxidoreductase purified from E. coli native membranes by Ni-NTA affinity chromatography.
Entity ID: #1-#4 / Source: NATURAL
Molecular weightValue: 0.144 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: C41
Buffer solutionpH: 8
Details: 20mM Tris-HCL, 150mM NaCl, 2% Glycerol, filtered and degassed.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSodium ChlorideNaClSodium chloride1
32 %GlycerolC3H8O31
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.1 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: 8050
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7PHENIXmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1277595
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45014 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 59.97 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1FFT

1fft


Accession code: 1FFT / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01210289
ELECTRON MICROSCOPYf_angle_d1.45713992
ELECTRON MICROSCOPYf_dihedral_angle_d16.6613503
ELECTRON MICROSCOPYf_chiral_restr0.071529
ELECTRON MICROSCOPYf_plane_restr0.0161674

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