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- EMDB-37895: PNPase mutant of Mycobacterium tuberculosis -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-37895
TitlePNPase mutant of Mycobacterium tuberculosis
Map data
Sample
  • Organelle or cellular component: PNPase of Mycobacterium tuberculosis
    • Protein or peptide: Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase
Keywordspolynucleotide phosphorylase / Mycobacterium tuberculosis / RNA degradation / TRANSFERASE
Function / homology:
Function and homology information
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsWang N / Sheng YN / Liu YT
Funding support China, 1 items
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 26, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37895.png

Downloads & links

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Map

FileDownload / File: emd_37895.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.9 Å
Density
Contour LevelBy AUTHOR: 3.7
Minimum - Maximum-15.839409 - 26.051476000000001
Average (Standard dev.)-0.000000000004737 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 198.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37895_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_37895_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 : PNPase of Mycobacterium tuberculosis

EntireName: PNPase of Mycobacterium tuberculosis
Components
  • Organelle or cellular component: PNPase of Mycobacterium tuberculosis
    • Protein or peptide: Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase

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Supramolecule #1: PNPase of Mycobacterium tuberculosis

SupramoleculeName: PNPase of Mycobacterium tuberculosis / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Macromolecule #1: Bifunctional guanosine pentaphosphate synthetase/polyribonucleoti...

MacromoleculeName: Bifunctional guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase
type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 82.117 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MMSAAEIDEG VFETTATIDN GSFGTRTIRF ETGRLALQAA GAVVAYLDDD NMLLSATTAS KNPKEHFNF FPLTVDVEER MYAAGRIPGS FFRREGRPST DAILTCRLID RPLRPSFVDG LRNEIQIVVT ILSLDPGDLY D VLAINAAS ...String:
MGSSHHHHHH SSGLVPRGSH MMSAAEIDEG VFETTATIDN GSFGTRTIRF ETGRLALQAA GAVVAYLDDD NMLLSATTAS KNPKEHFNF FPLTVDVEER MYAAGRIPGS FFRREGRPST DAILTCRLID RPLRPSFVDG LRNEIQIVVT ILSLDPGDLY D VLAINAAS ASTQLGGLPF SGPIGGVRVA LIDGTWVGFP TVDQIERAVF DMVVAGRIVE GDVAIMMVEA EATENVVELV EG GAQAPTE SVVAAGLEAA KPFIAALCTA QQELADAAGK SGKPTVDFPV FPDYGEDVYY SVSSVATDEL AAALTIGGKA ERD QRIDEI KTQVVQRLAD TYEGREKEVG AALRALTKKL VRQRILTDHF RIDGRGITDI RALSAEVAVV PRAHGSALFE RGET QILGV TTLDMIKMAQ QIDSLGPETS KRYMHHYNFP PFSTGETGRV GSPKRREIGH GALAERALVP VLPSVEEFPY AIRQV SEAL GSNGSTSMGS VCASTLALLN AGVPLKAPVA GIAMGLVSDD IQVEGAVDGV VERRFVTLTD ILGAEDAFGD MDFKVA GTK DFVTALQLDT KLDGIPSQVL AGALEQAKDA RLTILEVMAE AIDRPDEMSP YAPRVTTIKV PVDKIGEVIG PKGKVIN AI TEETGAQISI EDDGTVFVGA TDGPSAQAAI DKINAIANPQ LPTVGERFLG TVVKTTDFGA FVSLLPGRDG LVHISKLG K GKRIAKVEDV VNVGDKLRVE IADIDKRGKI SLILVADEDS TAAATDAATV TS

UniProtKB: UNIPROTKB: A0A9Q6P703

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTrisTris(hydroxymethyl)aminomethane hydrochloride
150.0 mMNaclsodium chloride
5.0 mMDTT1,4-Dithiothreitol

Details: 50mM Tris, pH 8.0, 150 Nacl, 5mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: NITROGEN / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: OTHER
Details: The value given for _em_vitrification.instrument is CRYOSOL VITROJET. This is not in a list of allowed values {'OTHER', 'FEI VITROBOT MARK IV', 'FEI VITROBOT MARK III', 'LEICA EM GP', 'LEICA ...Details: The value given for _em_vitrification.instrument is CRYOSOL VITROJET. This is not in a list of allowed values {'OTHER', 'FEI VITROBOT MARK IV', 'FEI VITROBOT MARK III', 'LEICA EM GP', 'LEICA KF80', 'GATAN CRYOPLUNGE 3', 'REICHERT-JUNG PLUNGER', 'SPOTITON', 'FEI VITROBOT MARK I', 'LEICA EM CPC', 'EMS-002 RAPID IMMERSION FREEZER', 'HOMEMADE PLUNGER', 'LEICA PLUNGER', 'FEI VITROBOT MARK II'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 3913 / Average exposure time: 1.6 sec. / Average electron dose: 2.19 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 217912
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION

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