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- PDB-6rx4: THE STRUCTURE OF BD OXIDASE FROM ESCHERICHIA COLI -

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Basic information

Entry
Database: PDB / ID: 6rx4
TitleTHE STRUCTURE OF BD OXIDASE FROM ESCHERICHIA COLI
Components(Cytochrome bd-I ubiquinol oxidase subunit ...) x 4
KeywordsOXIDOREDUCTASE / BD OXIDASE / TERMINAL OXIDASE
Function / homology
Function and homology information


quinol oxidase (electrogenic, proton-motive force generating) / oxidoreductase activity, acting on diphenols and related substances as donors / cytochrome complex / aerobic electron transport chain / outer membrane / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / oxidative phosphorylation / electron transfer activity / heme binding / membrane ...quinol oxidase (electrogenic, proton-motive force generating) / oxidoreductase activity, acting on diphenols and related substances as donors / cytochrome complex / aerobic electron transport chain / outer membrane / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / oxidative phosphorylation / electron transfer activity / heme binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
Cyd operon protein YbgT / Membrane bound YbgT-like / Membrane bound YbgT-like protein / Cytochrome ubiquinol oxidase subunit 1 / Cytochrome ubiquinol oxidase subunit 2 / Cytochrome bd terminal oxidase subunit I / Cytochrome bd terminal oxidase subunit II
Similarity search - Domain/homology
CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME B/C / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Ubiquinone-8 / Cytochrome bd-I ubiquinol oxidase subunit 1 / Cytochrome bd-I ubiquinol oxidase subunit 2 / Cytochrome bd-I ubiquinol oxidase subunit X
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRasmussen, T. / Boettcher, B. / Thesseling, A. / Friedrich, T.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation Germany
CitationJournal: Nat Commun / Year: 2019
Title: Homologous bd oxidases share the same architecture but differ in mechanism.
Authors: Alexander Theßeling / Tim Rasmussen / Sabrina Burschel / Daniel Wohlwend / Jan Kägi / Rolf Müller / Bettina Böttcher / Thorsten Friedrich /
Abstract: Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus ...Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
History
DepositionJun 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: 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.name

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Structure visualization

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Assembly

Deposited unit
A: Cytochrome bd-I ubiquinol oxidase subunit 1
B: Cytochrome bd-I ubiquinol oxidase subunit 2
C: Cytochrome bd-I ubiquinol oxidase subunit X
D: Cytochrome bd-I ubiquinol oxidase subunit Y
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,3529
Polymers107,0064
Non-polymers3,3465
Water1267
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16080 Å2
ΔGint-194 kcal/mol
Surface area31960 Å2
MethodPISA

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Components

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Cytochrome bd-I ubiquinol oxidase subunit ... , 4 types, 4 molecules ABCD

#1: Protein Cytochrome bd-I ubiquinol oxidase subunit 1 / Cytochrome bd-I oxidase subunit I / Cytochrome d ubiquinol oxidase subunit I


Mass: 58251.723 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria)
References: UniProt: P0ABJ9, quinol oxidase (electrogenic, proton-motive force generating)
#2: Protein Cytochrome bd-I ubiquinol oxidase subunit 2 / Cytochrome bd-I oxidase subunit II / Cytochrome d ubiquinol oxidase subunit II


Mass: 42479.828 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria)
References: UniProt: P0ABK2, quinol oxidase (electrogenic, proton-motive force generating)
#3: Protein/peptide Cytochrome bd-I ubiquinol oxidase subunit X / Cytochrome bd-I oxidase subunit X / Cytochrome d ubiquinol oxidase subunit X


Mass: 4043.663 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria)
References: UniProt: P56100, quinol oxidase (electrogenic, proton-motive force generating)
#4: Protein/peptide Cytochrome bd-I ubiquinol oxidase subunit Y / Cytochrome bd-I oxidase subunit Y / Cytochrome d ubiquinol oxidase subunit Y


Mass: 2230.741 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria)

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Non-polymers , 5 types, 12 molecules

#5: Chemical ChemComp-HEB / HEME B/C / HYBRID BETWEEN B AND C TYPE HEMES (PROTOPORPHYRIN IX CONTAINING FE)


Mass: 618.503 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H34FeN4O4
#6: Chemical ChemComp-HDD / CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME / Heme


Mass: 632.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O5
#7: Chemical ChemComp-PEE / 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 749.073 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H83NO8P / Comment: DOPE, phospholipid*YM
#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-octaen-1-yl]cyclohexa-2,5-diene-1,4-dione


Mass: 727.109 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C49H74O4
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O

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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: bd-oxidase from Escherichia coli / Type: COMPLEX
Details: Reconstruction into Amphipole A8-35 in the presence of the inhibitor aurachin C.
Entity ID: #1-#4 / Source: NATURAL
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMOPSC7H15NO4S1
220 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot time 3.5 sec, blot force 5

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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: 75000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 75 sec. / Electron dose: 59 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8663
Details: Images were collected in movie mode with 47 frames.

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Coot0.8.9.1model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.13.2998model refinement
Image processingDetails: Movies were motion corrected and dose weighted with the program Motioncorr2.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 197805 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 88 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 5DOQ

5doq

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