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- PDB-6rko: Cryo-EM structure of the E. coli cytochrome bd-I oxidase at 2.68 ... -

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Basic information

Entry
Database: PDB / ID: 6rko
TitleCryo-EM structure of the E. coli cytochrome bd-I oxidase at 2.68 A resolution
Components
  • (Cytochrome bd-I ubiquinol oxidase subunit ...) x 3
  • Uncharacterized protein YnhF
KeywordsMEMBRANE PROTEIN / Oxidoreductase Cytochrome bd oxidase bd oxidase 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 / cellular response to hypoxia / electron transfer activity / heme binding ...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 / cellular response to hypoxia / electron transfer activity / heme binding / metal ion binding / membrane / 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 / OXYGEN MOLECULE / Chem-POV / Ubiquinone-8 / Uncharacterized protein YnhF / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsSafarian, S. / Hahn, A. / Kuehlbrandt, W. / Michel, H.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research Germany
CitationJournal: Science / Year: 2019
Title: Active site rearrangement and structural divergence in prokaryotic respiratory oxidases.
Authors: S Safarian / A Hahn / D J Mills / M Radloff / M L Eisinger / A Nikolaev / J Meier-Credo / F Melin / H Miyoshi / R B Gennis / J Sakamoto / J D Langer / P Hellwig / W Kühlbrandt / H Michel /
Abstract: Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial ...Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.
History
DepositionApr 30, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / 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.identifier_ORCID / _citation_author.name
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.4May 22, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_validate_chiral
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: Cytochrome bd-I ubiquinol oxidase subunit 2
A: Cytochrome bd-I ubiquinol oxidase subunit 1
H: Uncharacterized protein YnhF
X: Cytochrome bd-I ubiquinol oxidase subunit X
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,44413
Polymers107,7754
Non-polymers5,6699
Water55831
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry, 1 x CydA 1 x CydB 1 x CydX 1 x CydH 2 x HEB 1 x HDD 1 x O2 1 x UQ8 31 x H2O 4 x POV
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15100 Å2
ΔGint-169 kcal/mol
Surface area30020 Å2
MethodPISA

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Components

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Cytochrome bd-I ubiquinol oxidase subunit ... , 3 types, 3 molecules BAX

#1: 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 genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: cydB, cyd-2, b0734, JW0723 / Production host: Escherichia coli (E. coli) / Strain (production host): C43 / Variant (production host): delta bo3
References: UniProt: P0ABK2, quinol oxidase (electrogenic, proton-motive force generating)
#2: 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 genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: cydA, cyd-1, b0733, JW0722 / Production host: Escherichia coli (E. coli) / Strain (production host): C43 / Variant (production host): delta bo3
References: UniProt: P0ABJ9, quinol oxidase (electrogenic, proton-motive force generating)
#4: 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 genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: cydX, ybgT, b4515, JW0724 / Production host: Escherichia coli (E. coli) / Strain (production host): C43 / Variant (production host): delta bo3
References: UniProt: P56100, quinol oxidase (electrogenic, proton-motive force generating)

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Protein/peptide , 1 types, 1 molecules H

#3: Protein/peptide Uncharacterized protein YnhF


Mass: 2999.651 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: ynhF, b4602, JW1649.1 / Production host: Escherichia coli (E. coli) / Strain (production host): C43 / Variant (production host): delta bo3 / References: UniProt: A5A618

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Non-polymers , 6 types, 40 molecules

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


Mass: 760.076 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#7: Chemical ChemComp-HDD / CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME


Mass: 632.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O5
#8: 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
#9: Chemical ChemComp-OXY / OXYGEN MOLECULE


Mass: 31.999 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: O2
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 31 / 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: Cytochrome bd-I oxidase from E. coli / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.1077 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43 / Plasmid: pet17b_cydABX-StrepII
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHepes1
2100 mMSodium chloride1
SpecimenConc.: 7 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Calibrated magnification: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2500 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 / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5463

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7Coot0.8model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.14model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3500000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 170000 / Algorithm: BACK PROJECTION / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 120 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient

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