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- EMDB-21875: ClpP and ClpX IGF loop in ClpX-ClpP complex bound to ssrA tagged GFP -

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Basic information

Entry
Database: EMDB / ID: EMD-21875
TitleClpP and ClpX IGF loop in ClpX-ClpP complex bound to ssrA tagged GFP
Map dataPhenix sharpened map.
Sample
  • Complex: ClpX-ClpP-GFPssrA-ATP/ATPrS
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
  • Ligand: water
KeywordsProtein degradation / AAA+ protease complex / CHAPERONE
Function / homology
Function and homology information


protein denaturation / HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / protein unfolding / serine-type peptidase activity ...protein denaturation / HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / protein unfolding / serine-type peptidase activity / proteasomal protein catabolic process / proteolysis involved in protein catabolic process / ATP-dependent protein folding chaperone / response to radiation / disordered domain specific binding / unfolded protein binding / ATPase binding / response to heat / protease binding / protein dimerization activity / cell division / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
Clp protease, ATP-binding subunit ClpX, bacteria / 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 ...Clp protease, ATP-binding subunit ClpX, bacteria / 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 ATP-binding subunit ClpX
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsFei X / Sauer RT
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM-101988 United States
CitationJournal: Elife / Year: 2020
Title: Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates.
Authors: Xue Fei / Tristan A Bell / Sarah R Barkow / Tania A Baker / Robert T Sauer /
Abstract: When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In and other eubacteria, the ...When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.
History
DepositionApr 29, 2020-
Header (metadata) releaseNov 4, 2020-
Map releaseNov 4, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wr2
  • Surface level: 7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21875.png

Downloads & links

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Map

FileDownload / File: emd_21875.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhenix sharpened map.
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 4.07 / Movie #1: 4.07
Minimum - Maximum-28.055541999999999 - 43.423428000000001
Average (Standard dev.)-0.00000000000209 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 348.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z348.000348.000348.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-28.05643.423-0.000

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Supplemental data

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Sample components

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Entire : ClpX-ClpP-GFPssrA-ATP/ATPrS

EntireName: ClpX-ClpP-GFPssrA-ATP/ATPrS
Components
  • Complex: ClpX-ClpP-GFPssrA-ATP/ATPrS
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
  • Ligand: water

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Supramolecule #1: ClpX-ClpP-GFPssrA-ATP/ATPrS

SupramoleculeName: ClpX-ClpP-GFPssrA-ATP/ATPrS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli) / Strain: ER2566

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

MacromoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpX / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 42.355812 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH DYDIPTTENL YFQGSSALPT PHEIRNHLDD YVIGQEQAKK VLAVAVYNHY KRLRNGDTSN GVELGKSNIL LIGPTGSGK TLLAETLARL LDVPFTMADA TTLTEAGYVG EDVENIIQKL LQKSDYDVQK AQRGIVYIDE IDKISRKSDN P SITRDVSG ...String:
MGSSHHHHHH DYDIPTTENL YFQGSSALPT PHEIRNHLDD YVIGQEQAKK VLAVAVYNHY KRLRNGDTSN GVELGKSNIL LIGPTGSGK TLLAETLARL LDVPFTMADA TTLTEAGYVG EDVENIIQKL LQKSDYDVQK AQRGIVYIDE IDKISRKSDN P SITRDVSG EGVQQALLKL IEGTVAAVPP QGGRKHPQQE FLQVDTSKIL FICGGAFAGL DKVISHRVET GSGIGFGATV KA KSDKASE GELLAQVEPE DLIKFGLIPE FIGRLPVVAT LNELSEEALI QILKEPKNAL TKQYQALFNL EGVDLEFRDE ALD AIAKKA MARKTGARGL RSIVEAALLD TMYDLPSMED VEKVVIDESV IDGQSEPLLI YGKPEAQQAS GE

UniProtKB: ATP-dependent Clp protease ATP-binding subunit ClpX

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Macromolecule #2: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 23.468869 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: LVPMVIEQTS RGERSFDIYS RLLKERVIFL TGQVEDHMAN LIVAQMLFLE AENPEKDIYL YINSPGGVIT AGMSIYDTMQ FIKPDVSTI CMGQAASMGA FLLTAGAKGK RFCLPNSRVM IHQPLGGYQG QATDIEIHAR EILKVKGRMN ELMALHTGQS L EQIERDTE ...String:
LVPMVIEQTS RGERSFDIYS RLLKERVIFL TGQVEDHMAN LIVAQMLFLE AENPEKDIYL YINSPGGVIT AGMSIYDTMQ FIKPDVSTI CMGQAASMGA FLLTAGAKGK RFCLPNSRVM IHQPLGGYQG QATDIEIHAR EILKVKGRMN ELMALHTGQS L EQIERDTE RDRFLSAPEA VEYGLVDSIL THRNENLYFQ SLEHHHHHH

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 14 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
25.0 mMMgCl2
100.0 mMKCl
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Detailsrapid mixing of ClpX, ClpP, ATPrS with GFPssrA substrate and ATP.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2.5 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 4525 / Average exposure time: 60.0 sec. / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ppe

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
RELION (ver. 3.0.8)
PHENIX (ver. 1.14)map sharpening

Details: with multibody refinement / Number images used: 334069
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ppe


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6wr2:
ClpP and ClpX IGF loop in ClpX-ClpP complex bound to ssrA tagged GFP

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