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- PDB-7ose: cytochrome bd-II type oxidase with bound aurachin D -

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Basic information

Entry
Database: PDB / ID: 7ose
Titlecytochrome bd-II type oxidase with bound aurachin D
Components
  • (Cytochrome bd-II ubiquinol oxidase subunit ...) x 2
  • Putative cytochrome bd-II ubiquinol oxidase subunit AppX
KeywordsMEMBRANE PROTEIN / terminal oxidase / Q-loop / inhibitor binding
Function / homology
Function and homology information


ubiquinol oxidase (H+-transporting) / cytochrome bo3 ubiquinol oxidase activity / cytochrome complex / aerobic electron transport chain / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / cell outer membrane / electron transfer activity / heme binding / metal ion binding / membrane / plasma membrane
Similarity search - Function
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
Aurachin D / CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME B/C / Ubiquinone-8 / Putative cytochrome bd-II ubiquinol oxidase subunit AppX / Cytochrome bd-II ubiquinol oxidase subunit 2 / Cytochrome bd-II ubiquinol oxidase subunit 1
Similarity search - Component
Biological speciesEscherichia coli BW25113 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsGrauel, A. / Kaegi, J. / Rasmussen, T. / Wohlwend, D. / Boettcher, B. / Friedrich, T.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)278002225/RTG 2202 Germany
CitationJournal: Nat Commun / Year: 2021
Title: Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D.
Authors: Antonia Grauel / Jan Kägi / Tim Rasmussen / Iryna Makarchuk / Sabrina Oppermann / Aurélien F A Moumbock / Daniel Wohlwend / Rolf Müller / Frederic Melin / Stefan Günther / Petra Hellwig ...Authors: Antonia Grauel / Jan Kägi / Tim Rasmussen / Iryna Makarchuk / Sabrina Oppermann / Aurélien F A Moumbock / Daniel Wohlwend / Rolf Müller / Frederic Melin / Stefan Günther / Petra Hellwig / Bettina Böttcher / Thorsten Friedrich /
Abstract: Cytochrome bd quinol:O oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of ...Cytochrome bd quinol:O oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.
History
DepositionJun 8, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: 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
Revision 1.2Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Source and taxonomy / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / entity / entity_name_com / entity_src_gen / pdbx_initial_refinement_model / struct_ref / struct_ref_seq
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _entity.pdbx_description / _entity.pdbx_ec / _entity_name_com.name / _entity_src_gen.pdbx_gene_src_gene / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession

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Structure visualization

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Assembly

Deposited unit
A: Cytochrome bd-II ubiquinol oxidase subunit 1
B: Cytochrome bd-II ubiquinol oxidase subunit 2
C: Putative cytochrome bd-II ubiquinol oxidase subunit AppX
D: Cytochrome bd-II ubiquinol oxidase subunit 1
E: Cytochrome bd-II ubiquinol oxidase subunit 2
F: Putative cytochrome bd-II ubiquinol oxidase subunit AppX
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,94116
Polymers208,0216
Non-polymers5,92010
Water362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, mass photometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28540 Å2
ΔGint-336 kcal/mol
Surface area61070 Å2
MethodPISA

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Components

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Cytochrome bd-II ubiquinol oxidase subunit ... , 2 types, 4 molecules ADBE

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


Mass: 57962.469 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BW25113 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): DeltacyoABCD
References: UniProt: P26459, ubiquinol oxidase (H+-transporting)
#2: Protein Cytochrome bd-II ubiquinol oxidase subunit 2 / Cytochrome bd-II oxidase subunit II


Mass: 42448.543 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BW25113 (bacteria) / Gene: appB, cbdB, cyxB, b0979, JW0961 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): DeltacyoABCD
References: UniProt: P26458, ubiquinol oxidase (H+-transporting)

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Protein/peptide , 1 types, 2 molecules CF

#3: Protein/peptide Putative cytochrome bd-II ubiquinol oxidase subunit AppX


Mass: 3599.463 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BW25113 (bacteria) / Strain: K12 / Gene: appX, yccB, b4592, JW0961.1, b0979.1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): DeltacyoABCD / References: UniProt: P24244

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Non-polymers , 5 types, 12 molecules

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


Mass: 618.503 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H34FeN4O4
#5: Chemical ChemComp-HDD / CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME


Mass: 632.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O5
#6: Chemical ChemComp-0NI / Aurachin D / 2-methyl-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]-1H-quinolin-4-one


Mass: 363.536 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C25H33NO
#7: 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.: 2 / Source method: obtained synthetically / Formula: C49H74O4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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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 bd-II ubiquinol oxidase / Type: COMPLEX
Details: dimer of heterotrimers solubilised in amphipol A8-35 in complex with the inhibitor Aurachin D
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.208 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMOPSC7H15NO4S1
220 mMsodium chlorideNaCl1
3160 uMAurachin DC25H33NO1
SpecimenConc.: 5.6 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: 45 sec incubation, 6.5 sec blotting

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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: 75000 X / Nominal defocus max: 2400 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: 79 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1836

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
2RELION3.1particle selection
3EPUimage acquisition
5CTFFIND4.1CTF correction
8Coot0.8.9model fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX19-4092model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 800000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125497 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 94 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: WEIGHTED MAP SUM AT ATOM CENTERS
Atomic model buildingPDB-ID: 6RX4

6rx4


Accession code: 6RX4 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315368
ELECTRON MICROSCOPYf_angle_d0.68421036
ELECTRON MICROSCOPYf_dihedral_angle_d11.5365372
ELECTRON MICROSCOPYf_chiral_restr0.0392288
ELECTRON MICROSCOPYf_plane_restr0.0032608

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