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- EMDB-20051: CryoEM focus classification map of the hyperactive ClpB mutant K4... -

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Basic information

Entry
Database: EMDB / ID: EMD-20051
TitleCryoEM focus classification map of the hyperactive ClpB mutant K476C, bound to casein, NTD-trimer
Map data
Samplehyperactive ClpB mutant K476C bound to casein
  • hyperactive ClpB mutant K476C_NTD trimer
  • alpha-s1-casein
  • Hyperactive disaggregase ClpB
  • Alpha S1-casein
Function / homology
Function and homology information


protein metabolic process / response to unfolded protein / ATPase activity, coupled / response to heat / protein refolding / membrane / ATP binding / identical protein binding / cytosol
ClpA/B, conserved site 1 / Chaperonin ClpB / AAA domain (Cdc48 subfamily) / Clp amino terminal domain, pathogenicity island component / ATPase family associated with various cellular activities (AAA) / ClpA/ClpB, AAA lid domain / Clp, N-terminal domain superfamily / ClpA/B, conserved site 2 / P-loop containing nucleoside triphosphate hydrolase / Clp ATPase, C-terminal ...ClpA/B, conserved site 1 / Chaperonin ClpB / AAA domain (Cdc48 subfamily) / Clp amino terminal domain, pathogenicity island component / ATPase family associated with various cellular activities (AAA) / ClpA/ClpB, AAA lid domain / Clp, N-terminal domain superfamily / ClpA/B, conserved site 2 / P-loop containing nucleoside triphosphate hydrolase / Clp ATPase, C-terminal / Chaperonins clpA/B signature 1. / Clp, N-terminal / AAA lid domain / ATPase, AAA-type, core / AAA+ ATPase domain / ClpA/B family / Chaperonins clpA/B signature 2. / C-terminal, D2-small domain, of ClpB protein
Chaperone protein ClpB
Biological speciesEscherichia coli K-12 (bacteria) / Bos taurus (cattle) / Bovine (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsRizo AR / Lin J-B / Gates SN / Tse E / Bart SM / Castellano LM / Dimaio F / Shorter J / Southworth DR
CitationJournal: Nat Commun / Year: 2019
Title: Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.
Authors: Alexandrea N Rizo / JiaBei Lin / Stephanie N Gates / Eric Tse / Stephen M Bart / Laura M Castellano / Frank DiMaio / James Shorter / Daniel R Southworth /
Abstract: Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains ...Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.
Validation ReportPDB-ID: 6og3

SummaryFull reportAbout validation report
DateDeposition: Apr 1, 2019 / Header (metadata) release: May 1, 2019 / Map release: Jun 12, 2019 / Update: Jun 19, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0117
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0117
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6og3
  • Surface level: 0.0117
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20051.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 264.192 Å
1.03 Å/pix.
x 256 pix.
= 264.192 Å
1.03 Å/pix.
x 256 pix.
= 264.192 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.032 Å
Density
Contour LevelBy AUTHOR: 0.0117 / Movie #1: 0.0117
Minimum - Maximum-0.026774807 - 0.06934049
Average (Standard dev.)0.000086781474 (±0.0012235555)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 264.192 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0321.0321.032
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z264.192264.192264.192
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0270.0690.000

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Supplemental data

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Sample components

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Entire hyperactive ClpB mutant K476C bound to casein

EntireName: hyperactive ClpB mutant K476C bound to casein / Number of components: 5

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Component #1: protein, hyperactive ClpB mutant K476C bound to casein

ProteinName: hyperactive ClpB mutant K476C bound to casein / Recombinant expression: No

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Component #2: protein, hyperactive ClpB mutant K476C_NTD trimer

ProteinName: hyperactive ClpB mutant K476C_NTD trimer / Recombinant expression: No
SourceSpecies: Escherichia coli K-12 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, alpha-s1-casein

ProteinName: alpha-s1-casein
Details: Protein was purchased from Sigma-Aldrich. Purified from bovine milk.
Recombinant expression: No
SourceSpecies: Bos taurus (cattle)

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Component #4: protein, Hyperactive disaggregase ClpB

ProteinName: Hyperactive disaggregase ClpB / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 96.960367 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, Alpha S1-casein

ProteinName: Alpha S1-casein / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.783958 kDa
SourceSpecies: Bovine (cattle)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.5 mg/mL / pH: 7.4
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 56 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.6 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 93588
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body
Input PDB model: 1KHY
Chain ID: A
Output model

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