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- PDB-6zji: Cryo-EM structure of wild-type KatG from M. tuberculosis -

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Basic information

Entry
Database: PDB / ID: 6zji
TitleCryo-EM structure of wild-type KatG from M. tuberculosis
ComponentsCatalase-peroxidase
KeywordsMETAL BINDING PROTEIN / Heme / Peroxidase-Catalase
Function / homology
Function and homology information


catalase-peroxidase / catalase activity / hydrogen peroxide catabolic process / response to oxidative stress / heme binding / metal ion binding
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
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 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
DepositionJun 29, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
AP1: Catalase-peroxidase
BP1: Catalase-peroxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,6084
Polymers161,3752
Non-polymers1,2332
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Homodimer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11830 Å2
ΔGint-107 kcal/mol
Surface area49570 Å2
MethodPISA

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Components

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


Mass: 80687.609 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: katG, AYJ03_010035, ERS007661_00994, ERS013471_02729, ERS024276_01596, ERS075361_01376, ERS094182_01139, F6W99_00474, FRD82_12135
Production host: Escherichia coli (E. coli) / References: UniProt: A0A0D5ZBI4, catalase-peroxidase
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 2 / 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 WT KatG from M. tuberculosis / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 161210 kDa/nm / Experimental value: YES
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
Specimen supportGrid material: COPPER
VitrificationCryogen name: ETHANE / Humidity: 95 %

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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: 50.29 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc7_3834refinement
PHENIX1.18rc7_3834refinement
EM software
IDNameVersionCategory
1Warp1.0.7particle selection
4cryoSPARCCTF correction
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96044 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 2CCA

2cca

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 146.75 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004611097
ELECTRON MICROSCOPYf_angle_d0.601415156
ELECTRON MICROSCOPYf_chiral_restr0.04161597
ELECTRON MICROSCOPYf_plane_restr0.0041982
ELECTRON MICROSCOPYf_dihedral_angle_d15.78151519

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