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- EMDB-1544: Structure of a chaperonin complex with non-native substrate prote... -

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Basic information

Entry
Database: EMDB / ID: EMD-1544
TitleStructure of a chaperonin complex with non-native substrate protein gp23
Map dataVolume contains GroEL complexed with denatured gp23 without imposed symmetry
Sample
  • Sample: GroEL-gp23
  • Protein or peptide: GroEL
  • Protein or peptide: gp23
Keywordschaperonin / bacteriophage T4 / capsid protein gp23 / GroEL
Function / homologyCapsid protein, T4-like bacteriophage-like / protein binding / Chaperonin Cpn60/GroEL
Function and homology information
Biological speciesEscherichia coli (E. coli) / Enterobacteria phage T4 (virus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 10.7 Å
AuthorsClare DK / Bakkes PJ / van Heerikhuizen H / van der Vies SM / Saibil HR
CitationJournal: Nature / Year: 2009
Title: Chaperonin complex with a newly folded protein encapsulated in the folding chamber.
Authors: D K Clare / P J Bakkes / H van Heerikhuizen / S M van der Vies / H R Saibil /
Abstract: A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, ...A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.
History
DepositionSep 2, 2008-
Header (metadata) releaseSep 3, 2008-
Map releaseApr 2, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1grl
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1544.gif

Downloads & links

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Map

FileDownload / File: emd_1544.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume contains GroEL complexed with denatured gp23 without imposed symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
2.8 Å/pix.
x 160 pix.
= 448. Å		2.8 Å/pix.
x 160 pix.
= 448. Å		2.8 Å/pix.
x 160 pix.
= 448. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.0902 / Movie #1: 0.2
Minimum - Maximum-1.76114 - 2.89855
Average (Standard dev.)0.00679069 (±0.0917656)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-81-81-81
Dimensions160160160
Spacing160160160
CellA=B=C: 448 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z448.000448.000448.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-81-81-81
NX/NY/NZ160160160
MAP C/R/S213
start NC/NR/NS-81-81-81
NC/NR/NS160160160
D min/max/mean-1.7612.8990.007

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Supplemental data

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Sample components

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Entire : GroEL-gp23

EntireName: GroEL-gp23
Components
  • Sample: GroEL-gp23
  • Protein or peptide: GroEL
  • Protein or peptide: gp23

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Supramolecule #1000: GroEL-gp23

SupramoleculeName: GroEL-gp23 / type: sample / ID: 1000
Details: gp23 was denatured in UREA and mixed with GroEL containing buffer
Oligomeric state: tetradecamer of GroEL bound to one copy of denatured gp23
Number unique components: 2
Molecular weightExperimental: 850 KDa / Theoretical: 850 KDa / Method: sequence

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Name.synonym: GroEL / Number of copies: 1 / Oligomeric state: tetradecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MC1009 / Location in cell: cytoplasm
Molecular weightExperimental: 56 KDa / Theoretical: 56 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pSL6
SequenceGO: protein binding / InterPro: Chaperonin Cpn60/GroEL

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Macromolecule #2: gp23

MacromoleculeName: gp23 / type: protein_or_peptide / ID: 2 / Name.synonym: Major capsid protein / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage T4 (virus) / synonym: Bacteriophage T4 / Location in cell: cytoplasmic
Molecular weightExperimental: 56 KDa / Theoretical: 56 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET2331
SequenceInterPro: Capsid protein, T4-like bacteriophage-like

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5 / Details: 40 mM Tris-HCl pH 7.5, 10 mM KCl, 10 mM MgCl2
StainingType: NEGATIVE / Details: Cryo
GridDetails: 400 mesh R2/2 c-flat grids
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: manual plunger / Method: blot for 2-3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: corrected at 150,00 times
DateJun 1, 2006
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 1.4 µm / Number real images: 150 / Average electron dose: 15 e/Å2 / Od range: 0.5 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsNo symmetry was applied
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: No symmetry was applied to the reconstruction / Number images used: 5600
Final angle assignmentDetails: SPIDER:theta 78-90 degrees, phi 0-360 degrees. Mirror option was on.

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Atomic model buiding 1

Initial modelPDB ID:

1grl

SoftwareName: URO
DetailsProtocol: Individual domains fitted as rigid bodies. The domains were separately fitted in URO
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation coefficient

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