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- PDB-7abr: Cryo-EM structure of B. subtilis ClpC (DWB mutant) hexamer bound ... -

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Basic information

Entry
Database: PDB / ID: 7abr
TitleCryo-EM structure of B. subtilis ClpC (DWB mutant) hexamer bound to a substrate polypeptide
Components
  • Negative regulator of genetic competence ClpC/MecB
  • substrate polypeptide
KeywordsCHAPERONE / AAA+ protein / protein degradation
Function / homology
Function and homology information


establishment of competence for transformation / ATP hydrolysis activity / ATP binding
Similarity search - Function
UVR domain / UVR domain profile. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. ...UVR domain / UVR domain profile. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Negative regulator of genetic competence ClpC/MecB
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsMorreale, F.E. / Meinhart, A. / Haselbach, D. / Clausen, T.
Funding support Austria, 1items
OrganizationGrant numberCountry
Vienna Science and Technology Fund (WWTF)WWTF // LS17-29 Austria
CitationJournal: Cell / Year: 2022
Title: BacPROTACs mediate targeted protein degradation in bacteria.
Authors: Francesca E Morreale / Stefan Kleine / Julia Leodolter / Sabryna Junker / David M Hoi / Stepan Ovchinnikov / Anastasia Okun / Juliane Kley / Robert Kurzbauer / Lukas Junk / Somraj Guha / ...Authors: Francesca E Morreale / Stefan Kleine / Julia Leodolter / Sabryna Junker / David M Hoi / Stepan Ovchinnikov / Anastasia Okun / Juliane Kley / Robert Kurzbauer / Lukas Junk / Somraj Guha / David Podlesainski / Uli Kazmaier / Guido Boehmelt / Harald Weinstabl / Klaus Rumpel / Volker M Schmiedel / Markus Hartl / David Haselbach / Anton Meinhart / Markus Kaiser / Tim Clausen /
Abstract: Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, ...Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.
History
DepositionSep 8, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 6, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Negative regulator of genetic competence ClpC/MecB
B: Negative regulator of genetic competence ClpC/MecB
C: Negative regulator of genetic competence ClpC/MecB
D: Negative regulator of genetic competence ClpC/MecB
E: Negative regulator of genetic competence ClpC/MecB
F: Negative regulator of genetic competence ClpC/MecB
S: substrate polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)554,73118
Polymers549,4727
Non-polymers5,25911
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area50360 Å2
ΔGint-79 kcal/mol
Surface area132110 Å2
MethodPISA

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Components

#1: Protein
Negative regulator of genetic competence ClpC/MecB


Mass: 91206.812 Da / Num. of mol.: 6 / Mutation: E280A, E618A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Strain: 168 / Gene: clpC, mecB, BSU00860 / Production host: Escherichia coli (E. coli) / References: UniProt: P37571
#2: Protein/peptide substrate polypeptide


Mass: 2230.741 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1B. subtilis ClpC (DWB mutant) hexamer bound to a substrate polypeptideCOMPLEX#1-#20RECOMBINANT
2Negative regulator of genetic competence ClpC/MecBCOMPLEX#11RECOMBINANT
3substrate polypeptideCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11
22NO
33NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Bacillus subtilis subsp. subtilis str. 168 (bacteria)224308
32Bacillus subtilis subsp. subtilis str. 168 (bacteria)224308
43unidentified (others)32644
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / C2 aperture diameter: 100 µm
Image recordingElectron dose: 54 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4455

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Processing

EM software
IDNameVersionCategory
1crYOLO1.3.5particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1034627
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212314 / Num. of class averages: 1 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 67.73 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010327546
ELECTRON MICROSCOPYf_angle_d0.962337188
ELECTRON MICROSCOPYf_chiral_restr0.05054329
ELECTRON MICROSCOPYf_plane_restr0.00454789
ELECTRON MICROSCOPYf_dihedral_angle_d26.12910646

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