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- PDB-6sfx: Cryo-EM structure of ClpP1/2 in the LmClpXP1/2 complex -

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Basic information

Entry
Database: PDB / ID: 6sfx
TitleCryo-EM structure of ClpP1/2 in the LmClpXP1/2 complex
Components(ATP-dependent Clp protease proteolytic subunit) x 2
KeywordsTRANSPORT PROTEIN / AAA+ / ATPase / Protease / Listeria / Motor protein / chaperone
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 / 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 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily ...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 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesListeria monocytogenes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsGatsogiannis, C. / Merino, F. / Raunser, S.
Funding support Germany, 3items
OrganizationGrant numberCountry
Max Planck Society Germany
European Research Council615984 Germany
German Research FoundationSFB1035 Germany
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Cryo-EM structure of the ClpXP protein degradation machinery.
Authors: Christos Gatsogiannis / Dora Balogh / Felipe Merino / Stephan A Sieber / Stefan Raunser /
Abstract: The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. ...The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP, where they are degraded. Formation of the complex involves a symmetry mismatch, because hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insight into the ClpX-ClpP cross-talk and activation mechanism. Comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of the complex mode of action of ClpXP and provides a structural framework for future pharmacological applications.
History
DepositionAug 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2020Group: Database references / Category: pdbx_database_related
Revision 1.2May 15, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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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)301,05514
Polymers301,05514
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area57670 Å2
ΔGint-243 kcal/mol
Surface area87550 Å2
MethodPISA

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Components

#1: Protein
ATP-dependent Clp protease proteolytic subunit / Endopeptidase Clp


Mass: 21384.139 Da / Num. of mol.: 7 / Mutation: S98A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes (bacteria)
Gene: clpP, AF298_10370, ARH36_13110, B1O28_12965, B1O52_13110, EFC24_09160, EHH22_02890, EK32_11400, JJ01_06855
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A3T2ER33, endopeptidase Clp
#2: Protein
ATP-dependent Clp protease proteolytic subunit / Endopeptidase Clp


Mass: 21623.789 Da / Num. of mol.: 7 / Mutation: S98A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes (bacteria) / Gene: clpP, LmNIHS28_02056 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A0B8R1W1, UniProt: Q9RQI6*PLUS, 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: LmClpXP1/2 heterocomplex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.7 / Experimental value: NO
Source (natural)Organism: Listeria monocytogenes (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
Details: The sample was crosslinked with 0.1% glutaraldehyde. The reaction was quenched after 30 s with 2 eq Tris-HCl.
Buffer component
IDConc.FormulaBuffer-ID
125 mMHEPES1
2200 mMKCl1
35 mMMgCl21
41 mMDTT1
50.5 mMATP1
65 %Glycerol1
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 %
Details: The sample was blotted after 45 s of incubation time

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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 / Nominal magnification: 59000 X / Calibrated magnification: 112807 X / Cs: 0 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 114 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3200
Details: Images were collected in movie mode at a frame rate of 50 ms

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Processing

EM software
IDNameVersionCategoryDetails
1SPHIRE1particle selectionParticles were picked with Cryolo
2EPUimage acquisition
4SPHIRE1CTF correctionCTER
7UCSF Chimeramodel fitting
9SPHIRE1initial Euler assignment
10SPHIRE1final Euler assignment
11SPHIRE1classification
12SPHIRE13D reconstruction
13Rosettamodel refinement
14NAMDmodel refinement
15PHENIXmodel refinement
16Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 613322
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 383927 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4RYF

4ryf


Accession code: 4RYF / Source name: PDB / Type: experimental model

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