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- PDB-8rgx: Cryo-EM structure of the Bacterial Proteasome Activator Bpa of My... -

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Basic information

Entry
Database: PDB / ID: 8rgx
TitleCryo-EM structure of the Bacterial Proteasome Activator Bpa of Mycobacterium tuberculosis
ComponentsBacterial proteasome activator
KeywordsCHAPERONE / protein degradation / quality control / proteasomal activator / mycobacteria
Function / homologyBacterial proteasome activator / Bacterial proteasome activator / proteasome accessory complex / positive regulation of proteasomal protein catabolic process / proteasome binding / peptidoglycan-based cell wall / protein homooligomerization / plasma membrane / Bacterial proteasome activator
Function and homology information
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZdanowicz, R. / von Rosen, T. / Afanasyev, P. / Boehringer, D. / Glockshuber, R. / Weber-Ban, E.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Commun / Year: 2025
Title: Substrates bind to residues lining the ring of asymmetrically engaged bacterial proteasome activator Bpa.
Authors: Tatjana von Rosen / Rafal Zdanowicz / Yasser El Hadeg / Pavel Afanasyev / Daniel Boehringer / Alexander Leitner / Rudi Glockshuber / Eilika Weber-Ban /
Abstract: Mycobacteria harbor a proteasome that was acquired by Actinobacteria through horizontal gene transfer and that supports the persistence of the human pathogen Mycobacterium tuberculosis within host ...Mycobacteria harbor a proteasome that was acquired by Actinobacteria through horizontal gene transfer and that supports the persistence of the human pathogen Mycobacterium tuberculosis within host macrophages. The core particle of the proteasome (20S CP) associates with ring-shaped activator complexes to degrade protein substrates. One of these is the bacterial proteasome activator Bpa that stimulates the ATP-independent proteasomal degradation of the heat shock repressor HspR. In this study, we determine the cryogenic electron microscopy 3D reconstruction of the complex between Bpa and its natural substrate HspR at 4.1 Å global resolution. The resulting maps allow us to identify regions of Bpa that interact with HspR. Using structure-guided site-directed mutagenesis and in vitro biochemical assays, we confirm the importance of the identified residues for Bpa-mediated substrate recruitment and subsequent proteasomal degradation. Additionally, we show that the dodecameric Bpa ring associates asymmetrically with the heptameric α-rings of the 20S CP, adopting a conformation resembling a hinged lid, while still engaging all seven docking sites on the proteasome.
History
DepositionDec 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 9, 2025Group: 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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bacterial proteasome activator
B: Bacterial proteasome activator
C: Bacterial proteasome activator
D: Bacterial proteasome activator
E: Bacterial proteasome activator
F: Bacterial proteasome activator
G: Bacterial proteasome activator
H: Bacterial proteasome activator
I: Bacterial proteasome activator
J: Bacterial proteasome activator
K: Bacterial proteasome activator
L: Bacterial proteasome activator


Theoretical massNumber of molelcules
Total (without water)239,08012
Polymers239,08012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26350 Å2
ΔGint-148 kcal/mol
Surface area55470 Å2
MethodPISA

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Components

#1: Protein
Bacterial proteasome activator


Mass: 19923.311 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: bpa / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WKX3
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: Bacterial Proteasome Activator Bpa of Mycobacterium tuberculosis
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE

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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 defocus max: 2600 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C12 (12 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229813 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5LFJ

5lfj


Accession code: 5LFJ / Details: Bacterial proteasome activator Bpa / Source name: PDB / Type: experimental model

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