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- PDB-7a2i: Cryo-EM structure of W107R KatG from M. tuberculosis -

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Basic information

Entry
Database: PDB / ID: 7a2i
TitleCryo-EM structure of W107R KatG from M. tuberculosis
ComponentsCatalase-peroxidase
KeywordsMETAL BINDING PROTEIN / Heme / Peroxidase-Catalase
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
PROTOPORPHYRIN IX CONTAINING FE / Catalase-peroxidase / Catalase-peroxidase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBlundell, T.L. / Chaplin, A.K. / Munir, A.
Funding support United Kingdom, 1items
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
DepositionAug 18, 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
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)161,9343
Polymers161,3172
Non-polymers6161
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6040 Å2
ΔGint-51 kcal/mol
Surface area47940 Å2
MethodPISA

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Components

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


Mass: 80658.594 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
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 W107R KatG from M. tuberculosis / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 161.1 kDa/nm / Experimental value: YES
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenConc.: 3 mg/ml / Embedding 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 FIELD
Image recordingElectron dose: 51.3 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 76067

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Processing

SoftwareName: PHENIX / Version: dev_3958: / Classification: refinement
EM software
IDNameCategory
4cryoSPARCCTF correction
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50043 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 2CCA

2cca


Accession code: 2CCA / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049440
ELECTRON MICROSCOPYf_angle_d0.5412874
ELECTRON MICROSCOPYf_dihedral_angle_d4.9751315
ELECTRON MICROSCOPYf_chiral_restr0.041387
ELECTRON MICROSCOPYf_plane_restr0.0041685

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