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- PDB-7fes: Cryo-EM structure of apo BsClpP at pH 4.2 -

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Basic information

Entry
Database: PDB / ID: 7fes
TitleCryo-EM structure of apo BsClpP at pH 4.2
ComponentsATP-dependent Clp protease proteolytic subunit
KeywordsHYDROLASE / ClpP / Bacillus subtilis
Function / homology
Function and homology information


endopeptidase Clp / endopeptidase Clp complex / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / ATPase binding / serine-type endopeptidase activity / identical protein binding / cytoplasm
Similarity search - Function
ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / ClpP/crotonase-like domain superfamily
Similarity search - Domain/homology
ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKim, L. / Lee, B.-G. / Kim, M.K. / Kwon, D.H. / Kim, H. / Brotz-Oesterhelt, H. / Roh, S.-H. / Song, H.K.
Funding support Korea, Republic Of, 6items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2020R1A2C3008285 Korea, Republic Of
National Research Foundation (NRF, Korea)2020R1A5A1019023 Korea, Republic Of
National Research Foundation (NRF, Korea)2021M3A9I4030068 Korea, Republic Of
National Research Foundation (NRF, Korea)2021M3A9I4021220 Korea, Republic Of
National Research Foundation (NRF, Korea)2019M3E5D6063871 Korea, Republic Of
National Research Foundation (NRF, Korea)2020R1A5A1018081 Korea, Republic Of
CitationJournal: EMBO J / Year: 2022
Title: Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis.
Authors: Leehyeon Kim / Byung-Gil Lee / Minki Kim / Min Kyung Kim / Do Hoon Kwon / Hyunmin Kim / Heike Brötz-Oesterhelt / Soung-Hun Roh / Hyun Kyu Song /
Abstract: The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. ...The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.
History
DepositionJul 21, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-dependent Clp protease proteolytic subunit
B: ATP-dependent Clp protease proteolytic subunit
C: ATP-dependent Clp protease proteolytic subunit
D: ATP-dependent Clp protease proteolytic subunit
E: ATP-dependent Clp protease proteolytic subunit
F: ATP-dependent Clp protease proteolytic subunit
G: ATP-dependent Clp protease proteolytic subunit
H: ATP-dependent Clp protease proteolytic subunit
I: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
L: ATP-dependent Clp protease proteolytic subunit
M: ATP-dependent Clp protease proteolytic subunit
N: ATP-dependent Clp protease proteolytic subunit


Theoretical massNumber of molelcules
Total (without water)313,66514
Polymers313,66514
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ATP-dependent Clp protease proteolytic subunit / Caseinolytic protease / Endopeptidase Clp / Stress protein G7


Mass: 22404.664 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: P80244, endopeptidase Clp

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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: Cryo-EM structure of apo BsClpP at pH 4.2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 4.2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium acetateC2H3NaO21
2500 mMpotassium chlorideKCl1
35 % (w/v)GlycerolC3H8O31
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: ATP-dependent Clp protease proteolytic subunit
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K

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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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 30 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM softwareName: cryoSPARC / Version: 2 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 341414
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29834 / Symmetry type: POINT

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