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- EMDB-21521: ClpAP Engaged2 State bound to RepA-GFP -

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Basic information

Entry
Database: EMDB / ID: EMD-21521
TitleClpAP Engaged2 State bound to RepA-GFP
Map dataClpAP Engaged2 State bound to RepA-GFP
Sample
  • Complex: ClpAP
    • Complex: ATP-dependent Clp protease ATP-binding subunit ClpA
      • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpA
    • Complex: ATP-dependent Clp protease proteolytic subunit (E.C.3.4.21.92)
      • Protein or peptide: RepA, green fluorescent protein fusion
    • Complex: RepA, green fluorescent protein fusion
      • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsAAA+ / Chaperone / Protease / Hsp100 / ATPase
Function / homology
Function and homology information


HslUV protease complex / endopeptidase Clp / endopeptidase Clp complex / 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 / proteasomal protein catabolic process / serine-type peptidase activity ...HslUV protease complex / endopeptidase Clp / endopeptidase Clp complex / 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 / proteasomal protein catabolic process / serine-type peptidase activity / 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 / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
ATP-dependent Clp protease ATP-binding subunit ClpA / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. ...ATP-dependent Clp protease ATP-binding subunit ClpA / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / Clp amino terminal domain, pathogenicity island component / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / 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 proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpA / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (E. coli) / synthetic construct (others) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLopez KL / Rizo AN
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
DepositionMar 4, 2020-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 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: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w21
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21521.png

Downloads & links

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Map

FileDownload / File: emd_21521.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClpAP Engaged2 State bound to RepA-GFP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 600 pix.
= 500.4 Å		0.83 Å/pix.
x 600 pix.
= 500.4 Å		0.83 Å/pix.
x 600 pix.
= 500.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.4805628 - 1.7085289
Average (Standard dev.)-0.0011302178 (±0.051474106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 500.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z500.400500.400500.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-0.4811.709-0.001

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Supplemental data

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Sample components

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Entire : ClpAP

EntireName: ClpAP
Components
  • Complex: ClpAP
    • Complex: ATP-dependent Clp protease ATP-binding subunit ClpA
      • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpA
    • Complex: ATP-dependent Clp protease proteolytic subunit (E.C.3.4.21.92)
      • Protein or peptide: RepA, green fluorescent protein fusion
    • Complex: RepA, green fluorescent protein fusion
      • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: ClpAP

SupramoleculeName: ClpAP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 840 KDa

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Supramolecule #2: ATP-dependent Clp protease ATP-binding subunit ClpA

SupramoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: ATP-dependent Clp protease proteolytic subunit (E.C.3.4.21.92)

SupramoleculeName: ATP-dependent Clp protease proteolytic subunit (E.C.3.4.21.92)
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #4: RepA, green fluorescent protein fusion

SupramoleculeName: RepA, green fluorescent protein fusion / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)

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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
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 84.321844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLNQELELSL NMAFARAREH RHEFMTVEHL LLALLSNPSA REALEACSVD LVALRQELEA FIEQTTPVLP ASEEERDTQP TLSFQRVLQ RAVFHVQSSG RNEVTGANVL VAIFSEQESQ AAYLLRKHEV SRLDVVNFIS HGTRKDEPTQ SSDPGSQPNS E EQAGGEER ...String:
MLNQELELSL NMAFARAREH RHEFMTVEHL LLALLSNPSA REALEACSVD LVALRQELEA FIEQTTPVLP ASEEERDTQP TLSFQRVLQ RAVFHVQSSG RNEVTGANVL VAIFSEQESQ AAYLLRKHEV SRLDVVNFIS HGTRKDEPTQ SSDPGSQPNS E EQAGGEER MENFTTNLNQ LARVGGIDPL IGREKELERA IQVLCRRRKN NPLLVGESGV GKTAIAEGLA WRIVQGDVPE VM ADCTIYS LDIGSLLAGT KYRGDFEKRF KALLKQLEQD TNSILFIDEI HTIIGAGAAS GGQVDAANLI KPLLSSGKIR VIG STTYQE FSNIFEKDRA LARRFQKIDI TEPSIEETVQ IINGLKPKYE AHHDVRYTAK AVRAAVELAV KYINDRHLPD KAID VIDEA GARARLMPVS KRKKTVNVAD IESVVARIAR IPEKSVSQSD RDTLKNLGDR LKMLVFGQDK AIEALTEAIK MARAG LGHE HKPVGSFLFA GPTGVGKTEV TVQLSKALGI ELLRFDMSEY MERHTVSRLI GAPPGYVGFD QGGLLTDAVI KHPHAV LLL DEIEKAHPDV FNILLQVMDN GTLTDNNGRK ADFRNVVLVM TTNAGVRETE RKSIGLIHQD NSTDAMEEIK KIFTPEF RN RLDNIIWFDH LSTDVIHQVV DKFIVELQVQ LDQKGVSLEV SQEARNWLAE KGYDRAMGAR PMARVIQDNL KKPLANEL L FGSLVDGGQV TVALDKEKNE LTYGFQSAQK HKAEAAH

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

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Macromolecule #2: RepA, green fluorescent protein fusion

MacromoleculeName: RepA, green fluorescent protein fusion / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.060531 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 3 / Number of copies: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.21265 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSYSGERDNF APHMALVPMV IEQTSRGERS FDIYSRLLKE RVIFLTGQVE DHMANLIVAQ MLFLEAENPE KDIYLYINSP GGVITAGMS IYDTMQFIKP DVSTICMGQA ASMGAFLLTA GAKGKRFCLP NSRVMIHQPL GGYQGQATDI EIHAREILKV K GRMNELMA ...String:
MSYSGERDNF APHMALVPMV IEQTSRGERS FDIYSRLLKE RVIFLTGQVE DHMANLIVAQ MLFLEAENPE KDIYLYINSP GGVITAGMS IYDTMQFIKP DVSTICMGQA ASMGAFLLTA GAKGKRFCLP NSRVMIHQPL GGYQGQATDI EIHAREILKV K GRMNELMA LHTGQSLEQI ERDTERDRFL SAPEAVEYGL VDSILTHRN

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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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

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*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
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
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 FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 39177
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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