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- EMDB-12451: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculos... -

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Basic information

Entry
Database: EMDB / ID: EMD-12451
TitleCryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
Map data
Sample
  • Complex: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
    • Protein or peptide: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit II) CydB (Cytochrome BD-I oxidase subunit II)
    • Protein or peptide: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit I) CydA (Cytochrome BD-I oxidase subunit I)
  • Ligand: OXYGEN MOLECULEAllotropes of oxygen
  • Ligand: MENAQUINONE-9Vitamin K2
  • Ligand: CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE
  • Ligand: HEME B/C
  • Ligand: water
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsSafarian S / Wu D / Krause KL / Michel H
Funding support Germany, New Zealand, 4 items
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, 2021-
Header (metadata) releaseSep 22, 2021-
Map releaseSep 22, 2021-
UpdateSep 29, 2021-
Current statusSep 29, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0239
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0239
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nkz
  • Surface level: 0.0239
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12451.png

Downloads & links

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Map

FileDownload / File: emd_12451.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.837 Å
Density
Contour LevelBy AUTHOR: 0.0239 / Movie #1: 0.0239
Minimum - Maximum-0.16093354 - 0.32472563
Average (Standard dev.)-0.00013626137 (±0.005778634)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 214.272 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8370.8370.837
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z214.272214.272214.272
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1610.325-0.000

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Supplemental data

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Mask #1

Fileemd_12451_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened Postprocess map

Fileemd_12451_additional_1.map
AnnotationSharpened Postprocess map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12451_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_12451_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of the cytochrome bd oxidase from M. tuberculos...

EntireName: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
Components
  • Complex: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
    • Protein or peptide: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit II) CydB (Cytochrome BD-I oxidase subunit II)
    • Protein or peptide: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit I) CydA (Cytochrome BD-I oxidase subunit I)
  • Ligand: OXYGEN MOLECULEAllotropes of oxygen
  • Ligand: MENAQUINONE-9Vitamin K2
  • Ligand: CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE
  • Ligand: HEME B/C
  • Ligand: water

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Supramolecule #1: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculos...

SupramoleculeName: Cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Recombinant plasmid: pYUB28b
Molecular weightExperimental: 91.5 KDa

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Macromolecule #1: Probable integral membrane cytochrome D ubiquinol oxidase (Subuni...

MacromoleculeName: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit II) CydB (Cytochrome BD-I oxidase subunit II)
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 37.650957 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MVLQELWFGV IAALFLGFFI LEGFDFGVGM LMAPFAHVGM GDPETHRRTA LNTIGPVWDG NEVWLITAGA AIFAAFPGWY ATVFSALYL PLLAILFGMI LRAVAIEWRG KIDDPKWRTG ADFGIAAGSW LPALLWGVAF AILVRGLPVD ANGHVALSIP D VLNAYTLL ...String:
MVLQELWFGV IAALFLGFFI LEGFDFGVGM LMAPFAHVGM GDPETHRRTA LNTIGPVWDG NEVWLITAGA AIFAAFPGWY ATVFSALYL PLLAILFGMI LRAVAIEWRG KIDDPKWRTG ADFGIAAGSW LPALLWGVAF AILVRGLPVD ANGHVALSIP D VLNAYTLL GGLATAGLFS LYGAVFIALK TSGPIRDDAY RFAVWLSLPV AGLVAGFGLW TQLAYGKDWT WLVLAVAGCA QA AATVLVW RRVSDGWAFM CTLIVVAAVV VLLFGALYPN LVPSTLNPQW SLTIHNASST PYTLKIMTWV TAFFAPLTVA YQT WTYWVF RQRISAERIP PPTGLARRAP

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Macromolecule #2: Probable integral membrane cytochrome D ubiquinol oxidase (Subuni...

MacromoleculeName: Probable integral membrane cytochrome D ubiquinol oxidase (Subunit I) CydA (Cytochrome BD-I oxidase subunit I)
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 53.863098 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MNVVDISRWQ FGITTVYHFI FVPLTIGLAP LIAVMQTLWV VTDNPAWYRL TKFFGKLFLI NFAIGVATGI VQEFQFGMNW SEYSRFVGD VFGAPLAMEG LAAFFFESTF IGLWIFGWNR LPRLVHLACI WIVAIAVNVS AFFIIAANSF MQHPVGAHYN P TTGRAELS ...String:
MNVVDISRWQ FGITTVYHFI FVPLTIGLAP LIAVMQTLWV VTDNPAWYRL TKFFGKLFLI NFAIGVATGI VQEFQFGMNW SEYSRFVGD VFGAPLAMEG LAAFFFESTF IGLWIFGWNR LPRLVHLACI WIVAIAVNVS AFFIIAANSF MQHPVGAHYN P TTGRAELS SIVVLLTNNT AQAAFTHTVS GALLTAGTFV AAVSAWWLVR SSTTHADSDT QAMYRPATIL GCWVALAATA GL LFTGDHQ GKLMFQQQPM KMASAESLCD TQTDPNFSVL TVGRQNNCDS LTRVIEVPYV LPFLAEGRIS GVTLQGIRDL QQE YQQRFG PNDYRPNLFV TYWSFRMMIG LMAIPVLFAL IALWLTRGGQ IPNQRWFSWL ALLTMPAPFL ANSAGWVFTE MGRQ PWVVV PNPTGDQLVR LTVKAGVSDH SATVVATSLL MFTLVYAVLA VIWCWLLKRY IVEGPLEHDA EPAAHGAPRD DEVAP LSFA Y

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Macromolecule #3: OXYGEN MOLECULE

MacromoleculeName: OXYGEN MOLECULE / type: ligand / ID: 3 / Number of copies: 1 / Formula: OXY
Molecular weightTheoretical: 31.999 Da
Chemical component information

ChemComp-O2:
OXYGEN MOLECULE / Allotropes of oxygen

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Macromolecule #4: MENAQUINONE-9

MacromoleculeName: MENAQUINONE-9 / type: ligand / ID: 4 / Number of copies: 1 / Formula: MQ9
Molecular weightTheoretical: 785.233 Da
Chemical component information

ChemComp-MQ9:
MENAQUINONE-9 / Vitamin K2

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Macromolecule #5: CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE

MacromoleculeName: CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / type: ligand / ID: 5 / Number of copies: 1 / Formula: HDD
Molecular weightTheoretical: 632.487 Da
Chemical component information

ChemComp-HDD:
CIS-HEME D HYDROXYCHLORIN GAMMA-SPIROLACTONE / Heme

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Macromolecule #6: HEME B/C

MacromoleculeName: HEME B/C / type: ligand / ID: 6 / Number of copies: 2 / Formula: HEB
Molecular weightTheoretical: 618.503 Da
Chemical component information

ChemComp-HEB:
HEME B/C

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 42 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7
Component:
ConcentrationNameFormula
20.0 mMHepes
100.0 mMNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 20.
DetailsMonodisperse sample of cytochrome bd oxidase reconstituted in lipid nanodiscs 1D1

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 96000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 12070 / Average exposure time: 5.0 sec. / Average electron dose: 15.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15000000
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 800000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 843799
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6rko

chain_id: A

6rko

chain_id: B
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 70 / Target criteria: Correlation coefficient
Output model

PDB-7nkz:
Cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 A resolution

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