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- EMDB-45652: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91 (... -

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Basic information

Entry
Database: EMDB / ID: EMD-45652
TitleComplex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91 (Local Refine Map of Dop-Pup91)
Map dataLocal refined map (sharpened)
Sample
  • Complex: Complex of M. smegmatis Dop with M. tuberculosis CoaX and Pup91
KeywordsPantothenate Kinase Isoform / CYTOSOLIC PROTEIN
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.12 Å
AuthorsChen J / 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)5R01AI174646-02 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
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_45652.png

Downloads & links

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Map

FileDownload / File: emd_45652.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refined 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: 0.2
Minimum - Maximum-0.8249088 - 1.5636798
Average (Standard dev.)0.00020677298 (±0.018859442)
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: Local refined map (halfA)

Fileemd_45652_half_map_1.map
AnnotationLocal refined map (halfA)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local refined map (halfB)

Fileemd_45652_half_map_2.map
AnnotationLocal refined map (halfB)
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

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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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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 reconstructionResolution.type: BY AUTHOR / Resolution: 2.12 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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