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- PDB-9fka: Cryo-EM structure of the reduced cytochrome bd oxidase from M. tu... -

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Basic information

Entry
Database: PDB / ID: 9fka
TitleCryo-EM structure of the reduced cytochrome bd oxidase from M. tuberculosis
Components(Probable integral membrane cytochrome D ubiquinol oxidase (Subunit ...) x 2
KeywordsOXIDOREDUCTASE / Ubiquinone / Demethylmenaquinone / Cryo-EM / Respiration / Substrate specificity / Disulfide regulation
Function / homology
Function and homology information


cytochrome complex / aerobic electron transport chain / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / electron transfer activity / heme binding / metal ion binding / plasma membrane
Similarity search - Function
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
Chem-CD4 / CARDIOLIPIN / CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / PROTOPORPHYRIN IX CONTAINING FE / MENAQUINONE-9 / PHOSPHATIDYLETHANOLAMINE / Probable integral membrane cytochrome D ubiquinol oxidase (Subunit I) CydA (Cytochrome BD-I oxidase subunit I) / Probable integral membrane cytochrome D ubiquinol oxidase (Subunit II) CydB (Cytochrome BD-I oxidase subunit II)
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsKayastha, K. / Bruenle, S.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Other government Netherlands
CitationJournal: J Biol Chem / Year: 2025
Title: Menaquinone-specific turnover by Mycobacterium tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond.
Authors: Tijn T van der Velden / Kanwal Kayastha / Caspar Y J Waterham / Steffen Brünle / Lars J C Jeuken /
Abstract: Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained interest as an antibiotic target because of its importance in survival under infectious conditions. Mtbd ...Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained interest as an antibiotic target because of its importance in survival under infectious conditions. Mtbd contains a characteristic disulfide bond that has been hypothesized to allow for Mtbd activity regulation at the enzymatic level, possibly helping M. tuberculosis to rapidly adapt to the hostile environment of the phagosome. Here, the role of the disulfide bond and quinone specificity have been determined by reconstitution of a minimal respiratory chain and the single-particle cryo-EM structure in the disulfide-reduced form. Mtbd was shown to be specific for menaquinone, while regulation by reduction of the Q-loop disulfide bond decreased oxidase activity up to 90%. Structural analysis shows that a salt bridge unique to Mtbd keeps the Q-loop partially structured in its disulfide-reduced form, which could facilitate the rapid activation of Mtbd upon exposure to reactive oxygen species. We signify Mtbd as the first redox sensory terminal oxidase and propose that this helps M. tuberculosis in the defense against reactive oxygen species encountered during infection.
History
DepositionJun 3, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit I) CydA (Cytochrome BD-I oxidase subunit I)
B: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit II) CydB (Cytochrome BD-I oxidase subunit II)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,6049
Polymers91,5142
Non-polymers6,0907
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14520 Å2
ΔGint-177 kcal/mol
Surface area29320 Å2

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Components

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Probable integral membrane cytochrome D ubiquinol oxidase (Subunit ... , 2 types, 2 molecules AB

#1: Protein Probable integral membrane cytochrome D ubiquinol oxidase (Subunit I) CydA (Cytochrome BD-I oxidase subunit I)


Mass: 53863.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: cydA, Rv1623c / Production host: Mycolicibacterium smegmatis (bacteria) / References: UniProt: L7N662
#2: Protein Probable integral membrane cytochrome D ubiquinol oxidase (Subunit II) CydB (Cytochrome BD-I oxidase subunit II)


Mass: 37650.957 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: cydB, Rv1622c / Production host: Mycolicibacterium smegmatis (bacteria) / References: UniProt: O06139

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Non-polymers , 6 types, 7 molecules

#3: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#4: Chemical ChemComp-MQ9 / MENAQUINONE-9


Mass: 785.233 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H80O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-HDD / CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME


Mass: 632.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O5 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C81H156O17P2 / Comment: phospholipid*YM
#8: Chemical ChemComp-CD4 / (2R,5R,11R,14R)-5,8,11-trihydroxy-5,11-dioxido-17-oxo-2,14-bis(tetradecanoyloxy)-4,6,10,12,16-pentaoxa-5,11-diphosphatriacont-1-yl tetradecanoate / tetramyristoyl-cardiolipin


Mass: 1241.633 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C65H126O17P2

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Details

Has ligand of interestY
Has protein modificationN

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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 / Type: COMPLEX
Details: Cytochrome BD-I oxidase bound with Heme B, cis-Heme D, Menaquinone-9, Cardiolipins, PE lipid
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.942 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis (bacteria)
Buffer solutionpH: 7.4 / Details: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.005% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-Hcl1
2150 mMSodium ChlorideNaCl1
30.005 %LMNG1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mAmp / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.04 sec. / Electron dose: 100 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 14078
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Objective aperture of 100 um / Energyfilter slit width: 20 eV
Image scansSampling size: 10 µm

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.0particle selection
2EPU2.1.0image acquisition
4cryoSPARC4.3.0CTF correction
7UCSF ChimeraX1.7.1model fitting
9UCSF ChimeraX1.7.1model refinement
10Coot0.89model refinement
11PHENIX1.21.1model refinement
13cryoSPARC4.3.0final Euler assignment
14cryoSPARC4.3.0classification
15cryoSPARC4.3.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 15300000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1277019 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Atomic model buildingPDB-ID: 7NKZ

7nkz


Accession code: 7NKZ / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.96 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036934
ELECTRON MICROSCOPYf_angle_d0.8589491
ELECTRON MICROSCOPYf_dihedral_angle_d13.4951164
ELECTRON MICROSCOPYf_chiral_restr0.0711024
ELECTRON MICROSCOPYf_plane_restr0.0041135

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