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- PDB-7nkz: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculos... -

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Basic information

Entry
Database: PDB / ID: 7nkz
TitleCryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
Components(Probable integral membrane cytochrome D ubiquinol oxidase (Subunit ...) x 2
KeywordsMEMBRANE PROTEIN / Terminal oxidase Oxidoreductase Oxygen reductase bd oxidase
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 / membrane => GO:0016020 / 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
CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / HEME B/C / MENAQUINONE-9 / OXYGEN MOLECULE / 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: 2.5 Å
AuthorsSafarian, S. / Wu, D. / Krause, K.L. / Michel, H.
Funding support Germany, New Zealand, 4items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
Max Planck SocietyNobel Laureate Fellowship Germany
Marsden Fund New Zealand
Royal Society of New ZealandCatalyst grant New Zealand
CitationJournal: Nat Commun / Year: 2021
Title: The cryo-EM structure of the bd oxidase from M. tuberculosis reveals a unique structural framework and enables rational drug design to combat TB.
Authors: Schara Safarian / Helen K Opel-Reading / Di Wu / Ahmad R Mehdipour / Kiel Hards / Liam K Harold / Melanie Radloff / Ian Stewart / Sonja Welsch / Gerhard Hummer / Gregory M Cook / Kurt L ...Authors: Schara Safarian / Helen K Opel-Reading / Di Wu / Ahmad R Mehdipour / Kiel Hards / Liam K Harold / Melanie Radloff / Ian Stewart / Sonja Welsch / Gerhard Hummer / Gregory M Cook / Kurt L Krause / Hartmut Michel /
Abstract: New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective ...New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.
History
DepositionFeb 19, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)94,2017
Polymers91,5142
Non-polymers2,6875
Water75742
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11310 Å2
ΔGint-138 kcal/mol
Surface area27710 Å2

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Components

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

#1: 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 MC2 155 (bacteria)
Strain (production host): mc2 155 / Variant (production host): delta cydAB / References: UniProt: O06139
#2: 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)
Strain: ATCC 25618 / H37Rv / Gene: cydA, Rv1623c
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain (production host): mc2 155 / Variant (production host): delta cydAB / References: UniProt: L7N662

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Non-polymers , 5 types, 47 molecules

#3: Chemical ChemComp-OXY / OXYGEN MOLECULE / Oxygen


Mass: 31.999 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: O2
#4: Chemical ChemComp-MQ9 / MENAQUINONE-9 / Vitamin K2


Mass: 785.233 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H80O2
#5: 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
#6: 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
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 42 / 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: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.0915 MDa / Experimental value: YES
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Plasmid: pYUB28b
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2100 mMNaClSodium chloride1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse sample of cytochrome bd oxidase reconstituted in lipid nanodiscs 1D1
Specimen supportDetails: 15 mA / Grid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force 20

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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 / Calibrated magnification: 96000 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 15 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 12070

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Processing

EM software
IDNameVersionCategory
2DigitalMicrographimage acquisition
3EPU2.9image acquisition
5CTFFINDCTF correction
8Coot0.8.9model fitting
10RELION3.1initial Euler assignment
12RELION3.1classification
14PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 15000000
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 843799 / Algorithm: EXACT BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 70 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16RKO

6rko

A1
26RKO

6rko

B1

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