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- PDB-7aa3: T275P after heme uptake from M. tuberculosis -

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Basic information

Entry
Database: PDB / ID: 7aa3
TitleT275P after heme uptake from M. tuberculosis
ComponentsCatalase-peroxidase
KeywordsMETAL BINDING PROTEIN / Heme / Catalase / Peroxidase enzyme
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 / peroxidase activity / peptidoglycan-based cell wall / hydrogen peroxide catabolic process ...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 / peroxidase activity / peptidoglycan-based cell wall / hydrogen peroxide catabolic process / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsBlundell, T.L. / Chaplin, A.K. / Munir, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation 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 3, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 18, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Catalase-peroxidase
B: Catalase-peroxidase


Theoretical massNumber of molelcules
Total (without water)161,3672
Polymers161,3672
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3430 Å2
ΔGint-22 kcal/mol
Surface area45120 Å2
MethodPISA

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Components

#1: Protein Catalase-peroxidase / / CP / Peroxidase/catalase


Mass: 80683.617 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: katG, ERS007661_00994, ERS013471_02729, ERS024276_01596, ERS075361_01376, ERS094182_01139, F6W99_00474, FRD82_12135
Production host: Escherichia coli (E. coli)
References: UniProt: A0A0D5ZBI4, UniProt: P9WIE5*PLUS, catalase-peroxidase

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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: Homodimer of KatG from M.tuberculosis / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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
Image recordingElectron dose: 50.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 165609 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048594
ELECTRON MICROSCOPYf_angle_d0.6211699
ELECTRON MICROSCOPYf_dihedral_angle_d17.7011179
ELECTRON MICROSCOPYf_chiral_restr0.0431277
ELECTRON MICROSCOPYf_plane_restr0.0041538

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