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- EMDB-11776: Cryo-EM structure of wild-type KatG from M. tuberculosis -

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Basic information

Entry
Database: EMDB / ID: EMD-11776
TitleCryo-EM structure of wild-type KatG from M. tuberculosis
Map data
Sample
  • Organelle or cellular component: Cryo-EM structure of WT KatG from M. tuberculosis
    • Protein or peptide: Catalase-peroxidase
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
KeywordsHeme / Peroxidase-Catalase / METAL BINDING PROTEIN
Function / homology
Function and homology information


oxidoreductase activity, acting on a heme group of donors, nitrogenous group as acceptor / Tolerance of reactive oxygen produced by macrophages / catalase-peroxidase / NADH binding / catalase activity / NADPH binding / positive regulation of DNA repair / peptidoglycan-based cell wall / hydrogen peroxide catabolic process / peroxidase activity ...oxidoreductase activity, acting on a heme group of donors, nitrogenous group as acceptor / Tolerance of reactive oxygen produced by macrophages / catalase-peroxidase / NADH binding / catalase activity / NADPH binding / positive regulation of DNA repair / peptidoglycan-based cell wall / hydrogen peroxide catabolic process / peroxidase activity / cellular response to hydrogen peroxide / response to oxidative stress / response to antibiotic / heme binding / extracellular region / metal ion binding / plasma membrane / cytosol
Similarity search - Function
Catalase-peroxidase haem / Peroxidases heam-ligand binding site / Peroxidase, active site / Peroxidases active site signature. / Plant heme peroxidase family profile. / Haem peroxidase / Peroxidase / Peroxidases proximal heme-ligand signature. / Haem peroxidase superfamily
Similarity search - Domain/homology
Catalase-peroxidase / Catalase-peroxidase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsBlundell TL / Chaplin AK
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationRG 86546 United Kingdom
CitationJournal: Structure / Year: 2021
Title: Using cryo-EM to understand antimycobacterial resistance in the catalase-peroxidase (KatG) from Mycobacterium tuberculosis.
Authors: Asma Munir / Michael T Wilson / Steven W Hardwick / Dimitri Y Chirgadze / Jonathan A R Worrall / Tom L Blundell / Amanda K Chaplin /
Abstract: Resolution advances in cryoelectron microscopy (cryo-EM) now offer the possibility to visualize structural effects of naturally occurring resistance mutations in proteins and also of understanding ...Resolution advances in cryoelectron microscopy (cryo-EM) now offer the possibility to visualize structural effects of naturally occurring resistance mutations in proteins and also of understanding the binding mechanisms of small drug molecules. In Mycobacterium tuberculosis the multifunctional heme enzyme KatG is indispensable for activation of isoniazid (INH), a first-line pro-drug for treatment of tuberculosis. We present a cryo-EM methodology for structural and functional characterization of KatG and INH resistance variants. The cryo-EM structure of the 161 kDa KatG dimer in the presence of INH is reported to 2.7 Å resolution allowing the observation of potential INH binding sites. In addition, cryo-EM structures of two INH resistance variants, identified from clinical isolates, W107R and T275P, are reported. In combination with electronic absorbance spectroscopy our cryo-EM approach reveals how these resistance variants cause disorder in the heme environment preventing heme uptake and retention, providing insight into INH resistance.
History
DepositionSep 21, 2020-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.596
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.596
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ag8
  • Surface level: 0.596
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11776.png

Downloads & links

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Map

FileDownload / File: emd_11776.map.gz / Format: CCP4 / Size: 93 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 290 pix.
= 240.7 Å		0.83 Å/pix.
x 290 pix.
= 240.7 Å		0.83 Å/pix.
x 290 pix.
= 240.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.596 / Movie #1: 0.596
Minimum - Maximum-1.5613085 - 2.9245842
Average (Standard dev.)0.0008934002 (±0.08577566)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions290290290
Spacing290290290
CellA=B=C: 240.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z290290290
origin x/y/z0.0000.0000.000
length x/y/z240.700240.700240.700
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS290290290
D min/max/mean-1.5612.9250.001

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Supplemental data

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Half map: #2

Fileemd_11776_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11776_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of WT KatG from M. tuberculosis

EntireName: Cryo-EM structure of WT KatG from M. tuberculosis
Components
  • Organelle or cellular component: Cryo-EM structure of WT KatG from M. tuberculosis
    • Protein or peptide: Catalase-peroxidase
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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Supramolecule #1: Cryo-EM structure of WT KatG from M. tuberculosis

SupramoleculeName: Cryo-EM structure of WT KatG from M. tuberculosis / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 161210 kDa/nm

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Macromolecule #1: Catalase-peroxidase

MacromoleculeName: Catalase-peroxidase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: catalase-peroxidase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 80.687609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPEQHPPITE TTTGAASNGC PVVGHMKYPV EGGGNQDWWP NRLNLKVLHQ NPAVADPMGA AFDYAAEVAT IDVDALTRDI EEVMTTSQP WWPADYGHYG PLFIRMAWHA AGTYRIHDGR GGAGGGMQRF APLNSWPDNA SLDKARRLLW PVKKKYGKKL S WADLIVFA ...String:
MPEQHPPITE TTTGAASNGC PVVGHMKYPV EGGGNQDWWP NRLNLKVLHQ NPAVADPMGA AFDYAAEVAT IDVDALTRDI EEVMTTSQP WWPADYGHYG PLFIRMAWHA AGTYRIHDGR GGAGGGMQRF APLNSWPDNA SLDKARRLLW PVKKKYGKKL S WADLIVFA GNCALESMGF KTFGFGFGRV DQWEPDEVYW GKEATWLGDE RYSGKRDLEN PLAAVQMGLI YVNPEGPNGN PD PMAAAVD IRETFRRMAM NDVETAALIV GGHTFGKTHG AGPADLVGPE PEAAPLEQMG LGWKSSYGTG TGKDAITSGI EVV WTNTPT KWDNSFLEIL YGYEWELTKS PAGAWQYTAK DGAGAGTIPD PFGGPGRSPT MLATDLSLRV DPIYERITRR WLEH PEELA DEFAKAWYKL IHRDMGPVAR YLGPLVPKQT LLWQDPVPAV SHDLVGEAEI ASLKSQIRAS GLTVSQLVST AWAAA SSFR GSDKRGGANG GRIRLQPQVG WEVNDPDGDL RKVIRTLEEI QESFNSAAPG NIKVSFADLV VLGGCAAIEK AAKAAG HNI TVPFTPGRTD ASQEQTDVES FAVLEPKADG FRNYLGKGNP LPAEYMLLDK ANLLTLSAPE MTVLVGGLRV LGANYKR LP LGVFTEASES LTNDFFVNLL DMGITWEPSP ADDGTYQGKD GSGKVKWTGS RVDLVFGSNS ELRALVEVYG ADDAQPKF V QDFVAAWDKV MNLDRFDVR

UniProtKB: Catalase-peroxidase

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Macromolecule #2: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 2 / Number of copies: 2 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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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

BufferpH: 7.2
VitrificationCryogen name: ETHANE / Chamber humidity: 95 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 48.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2cca

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 89703
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2cca


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-7ag8:
Cryo-EM structure of wild-type KatG from M. tuberculosis

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