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- EMDB-10162: Cryo-EM structure of the ClpXP1/2 protein degradation machinery -

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Basic information

Entry
Database: EMDB / ID: EMD-10162
TitleCryo-EM structure of the ClpXP1/2 protein degradation machinery
Map dataCryoEM density of ClpXP1/2 from Listeria monocytogenes. ClpP1/2 density filtered to 3.9A. ClpX density filtered to 6A.
Sample
  • Complex: LmClpXP1/2 heterocomplex
    • Complex: LmClpXP1/2
      • Protein or peptide: LmClpP1
      • Protein or peptide: LmClpP2
      • Protein or peptide: LmClpX
Function / homology
Function and homology information


endopeptidase Clp complex / endopeptidase Clp / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / protein catabolic process / unfolded protein binding / protein folding / peptidase activity / ATPase binding / protein dimerization activity ...endopeptidase Clp complex / endopeptidase Clp / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / protein catabolic process / unfolded protein binding / protein folding / peptidase activity / ATPase binding / protein dimerization activity / cell division / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding / cytoplasm
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 / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. ...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 / 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 / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ClpP/crotonase-like domain superfamily / 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 proteolytic subunit / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesListeria monocytogenes s (bacteria) / Listeria monocytogenes EGD (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsGatsogiannis C / Merino F / Raunser S
Funding support Germany, 3 items
OrganizationGrant numberCountry
Max Planck Society Germany
German Research FoundationSFB1035 Germany
European Research Council615984 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
DepositionJul 31, 2019-
Header (metadata) releaseOct 16, 2019-
Map releaseOct 16, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sfw, PDB-6sfx
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6sfw
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6sfx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10162.png

Downloads & links

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Map

FileDownload / File: emd_10162.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM density of ClpXP1/2 from Listeria monocytogenes. ClpP1/2 density filtered to 3.9A. ClpX density filtered to 6A.
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.08783696 - 0.1544804
Average (Standard dev.)0.0014495541 (±0.008608353)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 244.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z244.160244.160244.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0880.1540.001

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Supplemental data

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Half map: half map 1

Fileemd_10162_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 0

Fileemd_10162_half_map_2.map
Annotationhalf map 0
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : LmClpXP1/2 heterocomplex

EntireName: LmClpXP1/2 heterocomplex
Components
  • Complex: LmClpXP1/2 heterocomplex
    • Complex: LmClpXP1/2
      • Protein or peptide: LmClpP1
      • Protein or peptide: LmClpP2
      • Protein or peptide: LmClpX

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Supramolecule #1: LmClpXP1/2 heterocomplex

SupramoleculeName: LmClpXP1/2 heterocomplex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Listeria monocytogenes s (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #2: LmClpXP1/2

SupramoleculeName: LmClpXP1/2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Details: LmCLP1 and LmCLP2 assemble into two heptameric rings which stack face to face to form the LmClpP1/2 heterocomplex. The chaperone ClpX forms a hexamer, that associates with the LmClpP2 heptamer.
Source (natural)Organism: Listeria monocytogenes EGD (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: LmClpP1

MacromoleculeName: LmClpP1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Listeria monocytogenes EGD (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAENTKNENI TNILTQKLID TRTVLIYGEI NQELAEDVSK QLLLLESISN DPITIFINSQ GGHVEAGDTI HDMIKFIKPT VKVVGTGWV ASAGITIYLA AEKENRFSLP NTRYMIHQPA GGVQGQSTEI EIEAKEIIRM RERINRLIAE ATGQSYEQIS K DTDRNFWL ...String:
MAENTKNENI TNILTQKLID TRTVLIYGEI NQELAEDVSK QLLLLESISN DPITIFINSQ GGHVEAGDTI HDMIKFIKPT VKVVGTGWV ASAGITIYLA AEKENRFSLP NTRYMIHQPA GGVQGQSTEI EIEAKEIIRM RERINRLIAE ATGQSYEQIS K DTDRNFWL SVNEAKDYGI VNEIIENRDG LKMASWSHPQ FEK

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Macromolecule #2: LmClpP2

MacromoleculeName: LmClpP2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Listeria monocytogenes EGD (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNLIPTVIEQ TSRGERAYDI YSRLLKDRII MLGSAIDDNV ANSIVSQLLF LDAQDPEKDI FLYINSPGGS ISAGMAIYDT MNFVKADVQ TIGMGMAASM GSFLLTAGAN GKRFALPNAE IMIHQPLGGA QGQATEIEIA ARHILKIKER MNTIMAEKTG Q PYEVIARD ...String:
MNLIPTVIEQ TSRGERAYDI YSRLLKDRII MLGSAIDDNV ANSIVSQLLF LDAQDPEKDI FLYINSPGGS ISAGMAIYDT MNFVKADVQ TIGMGMAASM GSFLLTAGAN GKRFALPNAE IMIHQPLGGA QGQATEIEIA ARHILKIKER MNTIMAEKTG Q PYEVIARD TDRDNFMTAQ EAKDYGLIDD IIINKSGLKG HHHHHH DTD RNFWLSVNEA KDYGIVNEII ENRDGLKMAS WSHPQFEK

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Macromolecule #3: LmClpX

MacromoleculeName: LmClpX / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Listeria monocytogenes EGD (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MFKFNDEKGQ LKCSFCGKTQ DQVRKLVAGP GVYICDECIE LCNEIIEEEL GISEFVDFGE VPKPQEIRHI LSDYVIGQE RAKKALAVAV YNHYKRINSN ETKEDEVELS KSNICLIGPT GSGKTLLAQT LARILNVPFA I ADATSLTE AGYVGEDVEN ILLKLIQSAD ...String:
MFKFNDEKGQ LKCSFCGKTQ DQVRKLVAGP GVYICDECIE LCNEIIEEEL GISEFVDFGE VPKPQEIRHI LSDYVIGQE RAKKALAVAV YNHYKRINSN ETKEDEVELS KSNICLIGPT GSGKTLLAQT LARILNVPFA I ADATSLTE AGYVGEDVEN ILLKLIQSAD YDVEKAEKGI IYIDEIDKVA RKSENPSITR DVSGEGVQQA LL KILEGTV ASVPPQGGRK HPHQELIQID TGNILFIVGG AFDGIEQIVK NRMGEKVIGF GTDNAKLKDD ETY LSRVVP EDLLKFGLIP EFIGRLPVIA TLEQLDEAAL VSILTEPKNA LVKQYKRMLE LDDVELEFEP TALI EIAKE AIERKTGARG LRSIIEQIML EVMFEIPSRD DITKCIITEK AARGEEEPQL QLEDGSIIPI KTSA

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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

Concentration0.01 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormula
25.0 mMHEPES
200.0 mMKCl
5.0 mMMgCl2
1.0 mMDTT
0.5 mMATPAdenosine triphosphate
5.0 %Glycerol

Details: The sample was crosslinked with 0.1% glutaraldehyde. The reaction was quenched after 30sec with 2 eq. Tris-HCL.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3
Details: the sample was blotted after 45sec of incubation time..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 112807 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.0 mm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 3200 / Average exposure time: 2.0 sec. / Average electron dose: 114.0 e/Å2
Details: Images were collected in movie-mode at a frame rate of 50msec
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 613322
CTF correctionSoftware - Name: SPHIRE (ver. 1.0) / Software - details: CTER was used to estimate the CTF
Startup modelType of model: OTHER
Details: An initial model was computed from 2D class averages using SPHIRE's VIPER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.0)
Final 3D classificationSoftware - Name: SPHIRE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE (ver. 1.0) / Number images used: 383927

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

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

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

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