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- EMDB-20851: ClpA/ClpP Engaged State bound to RepA-GFP -

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Basic information

Entry
Database: EMDB / ID: EMD-20851
TitleClpA/ClpP Engaged State bound to RepA-GFP
Map data
Sample
  • Complex: ClpAP
    • Protein or peptide: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
    • Protein or peptide: RepA-GFP
  • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpA
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Function / homology
Function and homology information


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 ...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 / response to radiation / cellular response to heat / ATPase binding / response to heat / response to oxidative stress / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
ATP-dependent Clp protease ATP-binding subunit ClpA / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / 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 ...ATP-dependent Clp protease ATP-binding subunit ClpA / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / 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 / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily / ClpA/B family / 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 family associated with various cellular activities (AAA) / 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 ClpA / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLopez KL / Rizo AN / Southworth DR
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis.
Authors: Kyle E Lopez / Alexandrea N Rizo / Eric Tse / JiaBei Lin / Nathaniel W Scull / Aye C Thwin / Aaron L Lucius / James Shorter / Daniel R Southworth /
Abstract: The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ...The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop-substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.
History
DepositionOct 21, 2019-
Header (metadata) releaseApr 22, 2020-
Map releaseApr 22, 2020-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uqo
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20851.png

Downloads & links

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Map

FileDownload / File: emd_20851.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.853 Å
Density
Contour LevelBy AUTHOR: 0.65 / Movie #1: 0.65
Minimum - Maximum-1.5539625 - 3.5633495
Average (Standard dev.)0.00038190786 (±0.07746764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 511.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8530.8530.853
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z511.800511.800511.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-1.5543.5630.000

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Supplemental data

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Sample components

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Entire : ClpAP

EntireName: ClpAP
Components
  • Complex: ClpAP
    • Protein or peptide: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
    • Protein or peptide: RepA-GFP
  • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpA
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: ClpAP

SupramoleculeName: ClpAP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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

MacromoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpA / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 64.204504 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MENFTTNLNQ LARVGGIDPL IGREKELERA IQVLCRRRKN NPLLVGESGV GKTAIAEGLA WRIVQGDVPE VMADCTIYSL DIGSLLAGT KYRGDFEKRF KALLKQLEQD TNSILFIDEI HTIIGAGAAS GGQVDAANLI KPLLSSGKIR VIGSTTYQEF S NIFEKDRA ...String:
MENFTTNLNQ LARVGGIDPL IGREKELERA IQVLCRRRKN NPLLVGESGV GKTAIAEGLA WRIVQGDVPE VMADCTIYSL DIGSLLAGT KYRGDFEKRF KALLKQLEQD TNSILFIDEI HTIIGAGAAS GGQVDAANLI KPLLSSGKIR VIGSTTYQEF S NIFEKDRA LARRFQKIDI TEPSIEETVQ IINGLKPKYE AHHDVRYTAK AVRAAVELAV KYINDRHLPD KAIDVIDEAG AR ARLMPVS KRKKTVNVAD IESVVARIAR IPEKSVSQSD RDTLKNLGDR LKMLVFGQDK AIEALTEAIK MARAGLGHEH KPV GSFLFA GPTGVGKTEV TVQLSKALGI ELLRFDMSEY MERHTVSRLI GAPPGYVGFD QGGLLTDAVI KHPHAVLLLD EIEK AHPDV FNILLQVMDN GTLTDNNGRK ADFRNVVLVM TTNAGVRETE RKSIGLIHQD NSTDAMEEIK KIFTPEFRNR LDNII WFDH LSTDVIHQVV DKFIVELQVQ LDQKGVSLEV SQEARNWLAE KGYDRAMGAR PMARVIQDNL KKPLANELLF GSLVDG GQV TVALDKEKNE LTYGF

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Macromolecule #2: ATP-dependent Clp endopeptidase proteolytic subunit ClpP

MacromoleculeName: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
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: 21.472762 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: ALVPMVIEQT SRGERSFDIY SRLLKERVIF LTGQVEDHMA NLIVAQMLFL EAENPEKDIY LYINSPGGVI TAGMSIYDTM QFIKPDVST ICMGQAASMG AFLLTAGAKG KRFCLPNSRV MIHQPLGGYQ GQATDIEIHA REILKVKGRM NELMALHTGQ S LEQIERDT ...String:
ALVPMVIEQT SRGERSFDIY SRLLKERVIF LTGQVEDHMA NLIVAQMLFL EAENPEKDIY LYINSPGGVI TAGMSIYDTM QFIKPDVST ICMGQAASMG AFLLTAGAKG KRFCLPNSRV MIHQPLGGYQ GQATDIEIHA REILKVKGRM NELMALHTGQ S LEQIERDT ERDRFLSAPE AVEYGLVDSI LTHR

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Macromolecule #3: RepA-GFP

MacromoleculeName: RepA-GFP / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 783.958 Da
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 7 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 58616 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Average exposure time: 6.0 sec. / Average electron dose: 69.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 97000

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