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- EMDB-31559: Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-31559
TitleCryo-EM structure of BsClpP-ADEP1 complex at pH 6.5
Map data
Sample
  • Complex: Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5
    • Complex: BsClpP
      • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Complex: ADEP1
      • Protein or peptide: ADEP1
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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKim L / Lee B-G / Kim MK / Kwon DH / Kim H / Brotz-Oesterhelt H / Roh S-H / Song HK
Funding support Korea, Republic Of, 6 items
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, 2021-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateJul 20, 2022-
Current statusJul 20, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31559.png

Downloads & links

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Map

FileDownload / File: emd_31559.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 220 pix.
= 246.4 Å		1.12 Å/pix.
x 220 pix.
= 246.4 Å		1.12 Å/pix.
x 220 pix.
= 246.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-4.099952 - 6.4247704
Average (Standard dev.)0.006244569 (±0.2689881)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 246.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5

EntireName: Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5
Components
  • Complex: Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5
    • Complex: BsClpP
      • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Complex: ADEP1
      • Protein or peptide: ADEP1

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Supramolecule #1: Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5

SupramoleculeName: Cryo-EM structure of BsClpP-ADEP1 complex at pH 6.5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: BsClpP

SupramoleculeName: BsClpP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bacillus subtilis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: ADEP1

SupramoleculeName: ADEP1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 22.404664 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: NLIPTVIEQT NRGERAYDIY SRLLKDRIIM LGSAIDDNVA NSIVSQLLFL AAEDPEKEIS LYINSPGGSI TAGMAIYDTM QFIKPKVST ICIGMAASMG AFLLAAGEKG KRYALPNSEV MIHQPLGGAQ GQATEIEIAA KRILLLRDKL NKVLAERTGQ P LEVIERDT ...String:
NLIPTVIEQT NRGERAYDIY SRLLKDRIIM LGSAIDDNVA NSIVSQLLFL AAEDPEKEIS LYINSPGGSI TAGMAIYDTM QFIKPKVST ICIGMAASMG AFLLAAGEKG KRYALPNSEV MIHQPLGGAQ GQATEIEIAA KRILLLRDKL NKVLAERTGQ P LEVIERDT DRDNFKSAEE ALEYGLIDKI LTHTEDKKHH HHHH

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Macromolecule #2: ADEP1

MacromoleculeName: ADEP1 / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 736.855 Da
SequenceString:
(OTT)FSP(MAA)A(MP8)

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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: 6.5
Component:
ConcentrationFormulaName
50.0 mMC6H10N2O5ADA
200.0 mMNaClsodium chloride
5.0 % (w/v)C3H8O3Glycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 153078
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ktj

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 138976
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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