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- EMDB-1298: An expanded conformation of single-ring GroEL-GroES complex encap... -

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Basic information

Entry
Database: EMDB / ID: EMD-1298
TitleAn expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate.
Map dataThis is the surface representation of the electron density map at ~20-Angstrom resolution for the expanded conformation of SR398-AlphaBeta-GroES-Mg-ATP complex with significant substrate density inside the cavity
Sample
  • Sample: SR398-AlphaBeta-GroES-Mg-ATP
  • Protein or peptide: SR398
  • Protein or peptide: GroES
  • Protein or peptide: AlphaBeta
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsChen D-H / Song J-L / Chuang DT / Chiu W / Ludtke SJ
CitationJournal: Structure / Year: 2006
Title: An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate.
Authors: Dong-Hua Chen / Jiu-Li Song / David T Chuang / Wah Chiu / Steven J Ludtke /
Abstract: Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion ...Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with approximately 80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)(7) complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (alphabeta) assembly intermediate of mitochondrial branched-chain alpha-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both alpha and beta subunits of the substrate from tryptic digestion.
History
DepositionNov 18, 2006-
Header (metadata) releaseNov 18, 2006-
Map releaseNov 18, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.114899
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.114899
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1298.gif

Downloads & links

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Map

FileDownload / File: emd_1298.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the surface representation of the electron density map at ~20-Angstrom resolution for the expanded conformation of SR398-AlphaBeta-GroES-Mg-ATP complex with significant substrate density inside the cavity
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.17 Å/pix.
x 128 pix.
= 275.209 Å		2.17 Å/pix.
x 128 pix.
= 275.209 Å		2.17 Å/pix.
x 128 pix.
= 275.209 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.167 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 2.68 / Movie #1: 4.114899
Minimum - Maximum-3.8461 - 11.9573
Average (Standard dev.)-0.0182411 (±1.1714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 275.209 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1672.1672.167
M x/y/z127127127
origin x/y/z0.0000.0000.000
length x/y/z275.209275.209275.209
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-3.84611.957-0.018

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Supplemental data

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Sample components

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Entire : SR398-AlphaBeta-GroES-Mg-ATP

EntireName: SR398-AlphaBeta-GroES-Mg-ATP
Components
  • Sample: SR398-AlphaBeta-GroES-Mg-ATP
  • Protein or peptide: SR398
  • Protein or peptide: GroES
  • Protein or peptide: AlphaBeta

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Supramolecule #1000: SR398-AlphaBeta-GroES-Mg-ATP

SupramoleculeName: SR398-AlphaBeta-GroES-Mg-ATP / type: sample / ID: 1000
Details: Substrate AlphaBeta is an 86 kDa heterodimeric assembly intermediate of human mitochondrial branchedchain Alpha-ketoacid dehydrogenase (BCKD). GroEL/GroES is essential for promoting the ...Details: Substrate AlphaBeta is an 86 kDa heterodimeric assembly intermediate of human mitochondrial branchedchain Alpha-ketoacid dehydrogenase (BCKD). GroEL/GroES is essential for promoting the conversion of an otherwise kinetically trapped heterodimeric (AlphaBeta) intermediate to the native heterotetrameric (Alpha2Beta2) decarboxylase (E1) component of the human BCKD
Oligomeric state: GroES binds to SR398 ecapsulating substrate
Number unique components: 3
Molecular weightExperimental: 556 KDa / Theoretical: 556 KDa

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Macromolecule #1: SR398

MacromoleculeName: SR398 / type: protein_or_peptide / ID: 1 / Details: This is the GroEL protein with D398A mutation / Number of copies: 1 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E. coli / Cell: E. coli / Organelle: Cytoplasm / Location in cell: Cytoplasm
Molecular weightTheoretical: 400 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: PET

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Macromolecule #2: GroES

MacromoleculeName: GroES / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E. coli / Cell: E. coli / Organelle: Cytoplasm / Location in cell: Cytoplasm
Molecular weightTheoretical: 70 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pACYC

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Macromolecule #3: AlphaBeta

MacromoleculeName: AlphaBeta / type: protein_or_peptide / ID: 3
Details: Substrate AlphaBeta is an 86 kDa heterodimeric assembly intermediate of mitochondrial branchedchain Alpha-ketoacid dehydrogenase (BCKD). GroEL/GroES is essential for promoting the conversion ...Details: Substrate AlphaBeta is an 86 kDa heterodimeric assembly intermediate of mitochondrial branchedchain Alpha-ketoacid dehydrogenase (BCKD). GroEL/GroES is essential for promoting the conversion of an otherwise kinetically trapped heterodimeric (AlphaBeta) intermediate to the native heterotetrameric (Alpha2Beta2) decarboxylase (E1) component of the human mitochondrial BCKD
Number of copies: 2 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E. coli / Cell: Human mitochondria
Molecular weightTheoretical: 86 KDa
Recombinant expressionOrganism: Human mitochondria

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 50mM KPi, 150mM NaCl, 0.02% NaN3
GridDetails: 400 mesh quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: OTHER / Details: Vitrification instrument: FEI vitrobot
Method: Blot once and 3 seconds for each blot before plunging

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureAverage: 95 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification
DetailsThe magnification on CCD is 69220
DateSep 18, 2003
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 230 / Average electron dose: 18 e/Å2
Details: All images were recorded on a Gatan 4k by 4k 15-micron-per-pixel CCD
Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe particles were selected semi-automatically.
CTF correctionDetails: whole CCD frame
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 2520
Final angle assignmentDetails: EMAN
Final two d classificationNumber classes: 144

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