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- PDB-8wxf: PNPase of Mycobacterium tuberculosis -

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Basic information

Entry
Database: PDB / ID: 8wxf
TitlePNPase of Mycobacterium tuberculosis
ComponentsBifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase
KeywordsTRANSFERASE / polynucleotide phosphorylase / Mycobacterium tuberculosis / RNA degradation
Function / homology:
Function and homology information
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsWang, N. / Sheng, Y.N. / Liu, Y.T.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2021YFA1300904 China
CitationJournal: Arch Biochem Biophys / Year: 2024
Title: Cryo-EM structures of Mycobacterium tuberculosis polynucleotide phosphorylase suggest a potential mechanism for its RNA substrate degradation.
Authors: Na Wang / Yanan Sheng / Yutong Liu / Yaoting Guo / Jun He / Jinsong Liu /
Abstract: As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which ...As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.
History
DepositionOct 29, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase
B: Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase
C: Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase


Theoretical massNumber of molelcules
Total (without water)246,3543
Polymers246,3543
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase


Mass: 82117.984 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: gpsI, F6W99_01388 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A9Q6P703

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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: PNPase of Mycobacterium tuberculosis / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 50mM Tris, pH 8.0, 150 Nacl, 5mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris(hydroxymethyl)aminomethane hydrochlorideTris1
2150 mMsodium chlorideNacl1
35 mM1,4-DithiothreitolDTT1
SpecimenConc.: 6.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: protein sample was mixed with 15mM FOS-CHOLINE-8 (Anatrace) and rapidly loaded onto the holey carbon grids
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: CRYOSOL VITROJET / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 1.6 sec. / Electron dose: 2.22 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2981

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Processing

EM softwareName: RELION / Category: CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110060 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316626
ELECTRON MICROSCOPYf_angle_d0.64422566
ELECTRON MICROSCOPYf_dihedral_angle_d3.6752349
ELECTRON MICROSCOPYf_chiral_restr0.0442676
ELECTRON MICROSCOPYf_plane_restr0.0062979

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