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- EMDB-45654: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91 (... -

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Basic information

Entry
Database: EMDB / ID: EMD-45654
TitleComplex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91 (Composite Map)
Map dataComposite map (sharpened)
Sample
  • Complex: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
    • Protein or peptide: Type III pantothenate kinase
    • Protein or peptide: Pup deamidase/depupylase
    • Protein or peptide: Pup
  • Ligand: water
KeywordsPantothenate Kinase Isoform / CYTOSOLIC PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.04 Å
AuthorsChen J / Yoo JH / Ekiert DC / Bhabha G
Funding support United States, 2 items
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, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45654.png

Downloads & links

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Map

FileDownload / File: emd_45654.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map (sharpened)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 297. Å		0.83 Å/pix.
x 360 pix.
= 297. Å		0.83 Å/pix.
x 360 pix.
= 297. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 7.0
Minimum - Maximum-27.590498 - 47.473689999999998
Average (Standard dev.)0.0025280416 (±1.0304433)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Composite map (halfB)

Fileemd_45654_half_map_1.map
AnnotationComposite map (halfB)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Composite map (halfA)

Fileemd_45654_half_map_2.map
AnnotationComposite map (halfA)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91

EntireName: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
Components
  • Complex: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
    • Protein or peptide: Type III pantothenate kinase
    • Protein or peptide: Pup deamidase/depupylase
    • Protein or peptide: Pup
  • Ligand: water

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Supramolecule #1: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91

SupramoleculeName: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Macromolecule #1: Type III pantothenate kinase

MacromoleculeName: Type III pantothenate kinase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: pantothenate kinase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 29.341678 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLLAIDVRNT HTVVGLLSGM KEHAKVVQQW RIRTESEVTA DELALTIDGL IGEDSERLTG TAALSTVPSV LHEVRIMLDQ YWPSVPHVL IEPGVRTGIP LLVDNPKEVG ADRIVNCLAA YDRFRKAAIV VDFGSSICVD VVSAKGEFLG GAIAPGVQVS S DAAAARSA ...String:
MLLAIDVRNT HTVVGLLSGM KEHAKVVQQW RIRTESEVTA DELALTIDGL IGEDSERLTG TAALSTVPSV LHEVRIMLDQ YWPSVPHVL IEPGVRTGIP LLVDNPKEVG ADRIVNCLAA YDRFRKAAIV VDFGSSICVD VVSAKGEFLG GAIAPGVQVS S DAAAARSA ALRRVELARP RSVVGKNTVE CMQAGAVFGF AGLVDGLVGR IREDVSGFSV DHDVAIVATG HTAPLLLPEL HT VDHYDQH LTLQGLRLVF ERNLEVQRGR LKTAR

UniProtKB: Type III pantothenate kinase

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Macromolecule #2: Pup deamidase/depupylase

MacromoleculeName: Pup deamidase/depupylase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on peptide bonds (peptidases)
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 54.638484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQRIIGTEVE YGISSPSDPT ANPILTSTQA VLAYAAAAGI QRAKRTRWDY EVESPLRDAR GFDLSRSSGP PPIVDADEVG AANMILTNG ARLYVDHAHP EYSAPECTDP MDAVIWDKAG ERVMEAAARH VASVPGAAKL QLYKNNVDGK GASYGSHENY L MSRQTPFS ...String:
MQRIIGTEVE YGISSPSDPT ANPILTSTQA VLAYAAAAGI QRAKRTRWDY EVESPLRDAR GFDLSRSSGP PPIVDADEVG AANMILTNG ARLYVDHAHP EYSAPECTDP MDAVIWDKAG ERVMEAAARH VASVPGAAKL QLYKNNVDGK GASYGSHENY L MSRQTPFS AVIAGLTPFM VSRQVVTGSG RVGIGPSGDE PGFQLSQRAD YIEVEVGLET TLKRGIINTR DEPHADADKY RR LHVIIGD ANLAETSTYL KLGTTSLVLD LIEEGVDLSD LALARPVHAV HVISRDPSLR ATVALADGRE LTALALQRIY LDR VAKLVD SRDPDPRASH VIETWANVLD LLERDPMECA EILDWPAKLR LLEGFRQREN LTWQAPRLHL VDLQYSDVRL DKGL YNRLV ARGSMKRLVT EQQVLDAVEN PPTDTRAYFR GECLRRFGAD IAAASWDSVI FDLGGDSLVR IPTLEPLRGS KAHVG ALLD SVDSAVELVE QLTN

UniProtKB: Pup deamidase/depupylase

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Macromolecule #3: Pup

MacromoleculeName: Pup / type: protein_or_peptide / ID: 3 / Details: N-terminal TwinStrep tagged Pup91 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 7.465967 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MWSHPQFEKG GGSGGGSGGS AWSHPQFEKG SACELKLTEE TDDLLDEIDD VLEENAEDFV RAYVQKGGQ

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 130 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMNH2C(CH2OH)3tris(hydroxymethyl)aminomethane
150.0 mMNaClSodium Chloride
1.0 mMHSCH2CH(OH)CH(OH)CH2SH1,4-dithiothreitol
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 11015 / Average exposure time: 1.8 sec. / Average electron dose: 48.87 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8793538
Startup modelType of model: INSILICO MODEL
In silico model: Generated using Ab initio Reconstruction in cryoSPARC v4.5.1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.1) / Number images used: 2336294
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.1)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4.5.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation coefficient
Output model

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

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