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- PDB-9b78: Mycobacterium tuberculosis CoaX Homohexamer -

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Basic information

Entry
Database: PDB / ID: 9b78
TitleMycobacterium tuberculosis CoaX Homohexamer
ComponentsType III pantothenate kinase
KeywordsCYTOSOLIC PROTEIN / Pantothenate Kinase Isoform
Function / homology
Function and homology information


pantothenate kinase / pantothenate kinase activity / coenzyme A biosynthetic process / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Type III pantothenate kinase / Type III pantothenate kinase / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Type III pantothenate kinase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsChen, J. / Ekiert, D.C. / Bhabha, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA016087 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5R01AI174646-02 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Identification of a depupylation regulator for an essential enzyme in .
Authors: Shoshanna C Kahne / Jin Hee Yoo / James Chen / Kehilwe Nakedi / Lakshminarayan M Iyer / Gregory Putzel / Nora M Samhadaneh / Alejandro Pironti / L Aravind / Damian C Ekiert / Gira Bhabha / ...Authors: Shoshanna C Kahne / Jin Hee Yoo / James Chen / Kehilwe Nakedi / Lakshminarayan M Iyer / Gregory Putzel / Nora M Samhadaneh / Alejandro Pironti / L Aravind / Damian C Ekiert / Gira Bhabha / Kyu Y Rhee / K Heran Darwin /
Abstract: In , proteins that are posttranslationally modified with a prokaryotic ubiquitin-like protein (Pup) can be degraded by bacterial proteasomes. A single Pup-ligase and depupylase shape the pupylome, ...In , proteins that are posttranslationally modified with a prokaryotic ubiquitin-like protein (Pup) can be degraded by bacterial proteasomes. A single Pup-ligase and depupylase shape the pupylome, but the mechanisms regulating their substrate specificity are incompletely understood. Here, we identified a depupylation regulator, a protein called CoaX, through its copurification with the depupylase Dop. CoaX is a pseudopantothenate kinase that showed evidence of binding to pantothenate, an essential nutrient synthesizes, but not its phosphorylation. In a ∆ mutant, pantothenate synthesis enzymes including PanB, a substrate of the Pup-proteasome system (PPS), were more abundant than in the parental strain. In vitro, CoaX specifically accelerated depupylation of Pup~PanB, while addition of pantothenate inhibited this reaction. In culture, media supplementation with pantothenate decreased PanB levels, which required CoaX. Collectively, we propose CoaX regulates PanB abundance in response to pantothenate levels by modulating its vulnerability to proteolysis by proteasomes.
History
DepositionMar 27, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type III pantothenate kinase
B: Type III pantothenate kinase
C: Type III pantothenate kinase
D: Type III pantothenate kinase
E: Type III pantothenate kinase
F: Type III pantothenate kinase


Theoretical massNumber of molelcules
Total (without water)176,0506
Polymers176,0506
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
Type III pantothenate kinase / PanK-III / Pantothenic acid kinase


Mass: 29341.678 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: coaX, A4S10_03769, ERS007665_01672, ERS007679_02995, ERS007681_02234, ERS007688_03164, ERS007703_03054, ERS007741_04247, ERS027646_01657, ERS027659_02906, ERS053720_02846, GJE03_18915, SAMEA2683035_00698
Production host: Escherichia coli (E. coli) / References: UniProt: A0A045I4Z4, pantothenate kinase
Has protein modificationN

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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: Homohexameric assembly of M. tuberculosis CoaX / Type: COMPLEX / 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
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris(hydroxymethyl)aminomethaneNH2C(CH2OH)31
2150 mMSodium ChlorideNaCl1
31 mM1,4-dithiothreitolHSCH2CH(OH)CH(OH)CH2SH1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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 / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 48.51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7913
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.31particle selection
2Leginon3.6image acquisition
4cryoSPARC4.31CTF correction
7PHENIXv1.20.19model fitting
9cryoSPARC4.31initial Euler assignment
10cryoSPARC4.31final Euler assignment
11cryoSPARC4.31classification
12cryoSPARC4.313D reconstruction
13PHENIXv1.20.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8929726
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1285898 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingAccession code: P9WPA0 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212006
ELECTRON MICROSCOPYf_angle_d0.51116338
ELECTRON MICROSCOPYf_dihedral_angle_d11.2054332
ELECTRON MICROSCOPYf_chiral_restr0.0461980
ELECTRON MICROSCOPYf_plane_restr0.0052118

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