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- PDB-9cku: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91 -

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Basic information

Entry
Database: PDB / ID: 9cku
TitleComplex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
Components
  • Pup
  • Pup deamidase/depupylase
  • Type III pantothenate kinase
KeywordsCYTOSOLIC PROTEIN / Pantothenate Kinase Isoform
Function / homology
Function and homology information


Pup amidohydrolase / protein pupylation / pantothenate kinase / pantothenate kinase activity / hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides / coenzyme A biosynthetic process / Hydrolases; Acting on peptide bonds (peptidases) / proteasomal protein catabolic process / modification-dependent protein catabolic process / peptidase activity ...Pup amidohydrolase / protein pupylation / pantothenate kinase / pantothenate kinase activity / hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides / coenzyme A biosynthetic process / Hydrolases; Acting on peptide bonds (peptidases) / proteasomal protein catabolic process / modification-dependent protein catabolic process / peptidase activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Pup deamidase / Pup ligase/deamidase / Pup-ligase protein / Type III pantothenate kinase / Type III pantothenate kinase / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Type III pantothenate kinase / Pup deamidase/depupylase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Mycolicibacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.04 Å
AuthorsChen, J. / Yoo, J.H. / 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 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
DepositionJul 9, 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
G: Pup deamidase/depupylase
H: Pup


Theoretical massNumber of molelcules
Total (without water)238,1558
Polymers238,1558
Non-polymers00
Water2,342130
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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
#2: Protein Pup deamidase/depupylase / Deamidase of protein Pup


Mass: 54638.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria) / Gene: dop, MSMEG_3897, MSMEI_3807 / Production host: Escherichia coli (E. coli)
References: UniProt: A0QZ49, Hydrolases; Acting on peptide bonds (peptidases), Pup amidohydrolase
#3: Protein Pup


Mass: 7465.967 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal TwinStrep tagged Pup91 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Production host: Escherichia coli (E. coli)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 130 / Source method: isolated from a natural source / Formula: H2O
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: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
Type: COMPLEX / Entity ID: #1-#3 / 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.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
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: 48.87 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11015
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.1particle selection
4cryoSPARC4.5.1CTF correction
7PHENIXv1.20.19model fitting
9PHENIXv1.20.19model refinement
10cryoSPARC4.5.1initial Euler assignment
11cryoSPARC4.5.1final Euler assignment
12cryoSPARC4.5.1classification
13cryoSPARC4.5.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8793538
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2336294 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415415
ELECTRON MICROSCOPYf_angle_d0.66620966
ELECTRON MICROSCOPYf_dihedral_angle_d12.1985621
ELECTRON MICROSCOPYf_chiral_restr0.0682513
ELECTRON MICROSCOPYf_plane_restr0.0062728

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