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- PDB-6sfw: Cryo-EM Structure of the ClpX component of the ClpXP1/2 degradati... -

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Basic information

Entry
Database: PDB / ID: 6sfw
TitleCryo-EM Structure of the ClpX component of the ClpXP1/2 degradation machinery.
ComponentsATP-dependent Clp protease ATP-binding subunit ClpX
KeywordsTRANSPORT PROTEIN / AAA+ / ATPase / Protease / Listeria / Motor protein / chaperone
Function / homology
Function and homology information


protein catabolic process / unfolded protein binding / protein folding / peptidase activity / protein dimerization activity / cell division / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding
Similarity search - Function
Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein ...Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / 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, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent Clp protease ATP-binding subunit ClpX
Similarity search - Component
Biological speciesListeria monocytogenes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6 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

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Structure visualization

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Assembly

Deposited unit
O: ATP-dependent Clp protease ATP-binding subunit ClpX
P: ATP-dependent Clp protease ATP-binding subunit ClpX
Q: ATP-dependent Clp protease ATP-binding subunit ClpX
R: ATP-dependent Clp protease ATP-binding subunit ClpX
S: ATP-dependent Clp protease ATP-binding subunit ClpX
T: ATP-dependent Clp protease ATP-binding subunit ClpX


Theoretical massNumber of molelcules
Total (without water)278,9536
Polymers278,9536
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19520 Å2
ΔGint-92 kcal/mol
Surface area80270 Å2
MethodPISA

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Components

#1: Protein
ATP-dependent Clp protease ATP-binding subunit ClpX


Mass: 46492.172 Da / Num. of mol.: 6 / Mutation: E183Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes (bacteria)
Gene: clpX, ARJ20_08095, B4Y57_02215, D3B94_07125, DWE46_10580, DWE48_07885, FA029_07915, FORC68_1290, LmNIHS28_00678
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: L8DZH5, UniProt: Q8Y7K9*PLUS

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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: LmClpX / Type: COMPLEX
Details: Hexameric AAA+ ATPase that unfolds the substrate to be degraded by ClpP1/2
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 mMATPAdenosine triphosphate1
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 FIELDBright-field microscopy / 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 selectionCryolo
2EPUimage acquisition
4SPHIRE1CTF correctionCTER
7UCSF Chimeramodel fitting
9SPHIRE1initial Euler assignment
10SPHIRE1final Euler assignment
11SPHIRE1classification
12SPHIRE13D reconstruction
13NAMDmodel refinement
14Rosettamodel refinement
15PHENIXmodel refinement
16Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 613322
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 383927
Details: The overall resolution of the map is 4 A, but the ClpX region has significantly lower local resolution.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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