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- EMDB-1673: Cryo-EM reconstruction of the E.Coli ClpA ATPase -

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Basic information

Entry
Database: EMDB / ID: EMD-1673
TitleCryo-EM reconstruction of the E.Coli ClpA ATPase
Map dataClpA ATPase cryo EM reconstruction
Sample
  • Sample: E.Coli ClpA in complex with ClpP, both wild type
  • Protein or peptide: clpA
KeywordsProtease / ATPase / chaperone
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.6 Å
AuthorsEffantin G / DeDonatis GM / Ishikawa T / Maurizi MR / Steven AC
CitationJournal: Structure / Year: 2010
Title: Local and global mobility in the ClpA AAA+ chaperone detected by cryo-electron microscopy: functional connotations.
Authors: Grégory Effantin / Takashi Ishikawa / Gian Marco De Donatis / Michael R Maurizi / Alasdair C Steven /
Abstract: The ClpA chaperone combines with the ClpP peptidase to perform targeted proteolysis in the bacterial cytoplasm. ClpA monomer has an N-terminal substrate-binding domain and two AAA+ ATPase domains (D1 ...The ClpA chaperone combines with the ClpP peptidase to perform targeted proteolysis in the bacterial cytoplasm. ClpA monomer has an N-terminal substrate-binding domain and two AAA+ ATPase domains (D1 and D2). ClpA hexamers stack axially on ClpP heptamers to form the symmetry-mismatched protease. We used cryo-electron microscopy to visualize the ClpA-ATPgammaS hexamer, in the context of ClpAP complexes. Two segments lining the axial channel show anomalously low density, indicating that these motifs, which have been implicated in substrate translocation, are mobile. We infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier. The entire N domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15 residues were introduced into the linker, N domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. Based on these observations, we present a pseudo-atomic model of ClpAP holoenzyme, a dynamic proteolytic nanomachine.
History
DepositionJan 5, 2010-
Header (metadata) releaseJan 7, 2010-
Map releaseJun 1, 2010-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1673.png

Downloads & links

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Map

FileDownload / File: emd_1673.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClpA ATPase cryo EM reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.84 Å/pix.
x 120 pix.
= 220.8 Å		1.84 Å/pix.
x 120 pix.
= 220.8 Å		1.84 Å/pix.
x 120 pix.
= 220.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.2 / Movie #1: 5.2
Minimum - Maximum-8.102729999999999 - 11.872
Average (Standard dev.)0.140275 (±2.23425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-11900
Dimensions120120120
Spacing120120120
CellA=B=C: 220.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.841.841.84
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z220.800220.800220.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS0-1190
NC/NR/NS120120120
D min/max/mean-8.10311.8720.140

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Supplemental data

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Sample components

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Entire : E.Coli ClpA in complex with ClpP, both wild type

EntireName: E.Coli ClpA in complex with ClpP, both wild type
Components
  • Sample: E.Coli ClpA in complex with ClpP, both wild type
  • Protein or peptide: clpA

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Supramolecule #1000: E.Coli ClpA in complex with ClpP, both wild type

SupramoleculeName: E.Coli ClpA in complex with ClpP, both wild type / type: sample / ID: 1000
Oligomeric state: One homohexamer of ClpA binds to one tetradecamer of ClpP
Number unique components: 1
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa

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Macromolecule #1: clpA

MacromoleculeName: clpA / type: protein_or_peptide / ID: 1 / Name.synonym: clpA
Details: ClpA is in complex with ClpP but only ClpA was reconstructed
Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Organelle: Cytoplasm
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa

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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

Concentration0.12 mg/mL
BufferpH: 7.5 / Details: 150mM KCl, 50mM Tris-HCL,10mM MgCl2,1mM ATPgS
GridDetails: 300 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal magnification: 38000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 1.84 µm

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 8260
DetailsFocal pairs were used

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