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- EMDB-11708: Cryo-EM map of the activated B. subtilis ClpC arranged as a tetra... -

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Basic information

Entry
Database: EMDB / ID: EMD-11708
TitleCryo-EM map of the activated B. subtilis ClpC arranged as a tetramer of hexamers
Map data
Sample
  • Complex: Negative regulator of genetic competence ClpC/MecB
    • Protein or peptide: Negative regulator of genetic competence ClpC/MecB
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
Negative regulator of genetic competence ClpC/MecB
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsMorreale FE / Meinhart A / Haselbach D / Clausen T
Funding support Austria, 1 items
OrganizationGrant numberCountry
Other privateWWTF // 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, 2020-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateJul 6, 2022-
Current statusJul 6, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.36
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.36
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11708.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.058 Å
Density
Contour LevelBy AUTHOR: 0.36 / Movie #1: 0.36
Minimum - Maximum-0.2042477 - 0.87563694
Average (Standard dev.)0.00014580844 (±0.0733319)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 529.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0581.0581.058
M x/y/z500500500
origin x/y/z0.0000.0000.000
length x/y/z529.000529.000529.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS500500500
D min/max/mean-0.2040.8760.000

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Supplemental data

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Sample components

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Entire : Negative regulator of genetic competence ClpC/MecB

EntireName: Negative regulator of genetic competence ClpC/MecB
Components
  • Complex: Negative regulator of genetic competence ClpC/MecB
    • Protein or peptide: Negative regulator of genetic competence ClpC/MecB

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

SupramoleculeName: Negative regulator of genetic competence ClpC/MecB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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

MacromoleculeName: Negative regulator of genetic competence ClpC/MecB / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMFGRFTERA QKVLALAQEE ALRLGHNNIG TEHILLGLVR EGEGIAAKAL QALGLGSEKI QKEVESLIGR GQEMSQTIHY TPRAKKVIE LSMDEARKLG HSYVGTEHIL LGLIREGEGV AARVLNNLGV SLNKARQQVL QLLGSNETGS SAAGTNSNAN T PTLDSLAR ...String:
MMFGRFTERA QKVLALAQEE ALRLGHNNIG TEHILLGLVR EGEGIAAKAL QALGLGSEKI QKEVESLIGR GQEMSQTIHY TPRAKKVIE LSMDEARKLG HSYVGTEHIL LGLIREGEGV AARVLNNLGV SLNKARQQVL QLLGSNETGS SAAGTNSNAN T PTLDSLAR DLTAIAKEDS LDPVIGRSKE IQRVIEVLSR RTKNNPVLIG EPGVGKTAIA EGLAQQIINN EVPEILRDKR VM TLDMGTV VAGTKYRGEF EDRLKKVMDE IRQAGNIILF IDALHTLIGA GGAEGAIDAS NILKPSLARG ELQCIGATTL DEY RKYIEK DAALERRFQP IQVDQPSVDE SIQILQGLRD RYEAHHRVSI TDDAIEAAVK LSDRYISDRF LPDKAIDLID EAGS KVRLR SFTTPPNLKE LEQKLDEVRK EKDAAVQSQE FEKAASLRDT EQRLREQVED TKKSWKEKQG QENSEVTVDD IAMVV SSWT GVPVSKIAQT ETDKLLNMEN ILHSRVIGQD EAVVAVAKAV RRARAGLKDP KRPIGSFIFL GPTGVGKTEL ARALAE SIF GDEESMIRID MSEYMEKHST SRLVGSPPGY VGYDEGGQLT EKVRRKPYSV VLLDAIEKAH PDVFNILLQV LEDGRLT DS KGRTVDFRNT ILIMTSNVGA SELKRNKYVG FNVQDETQNH KDMKDKVMGE LKRAFRPEFI NRIDEIIVFH SLEKKHLT E IVSLMSDQLT KRLKEQDLSI ELTDAAKAKV AEEGVDLEYG ARPLRRAIQK HVEDRLSEEL LRGNIHKGQH IVLDVEDGE FVVKTTAKTN LEHHHHHH

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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

BufferpH: 7.5
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 4455 / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1173558
CTF correctionSoftware - Name: cryoSPARC (ver. 2)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 87575

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