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- EMDB-1297: An expanded conformation of single-ring GroEL-GroES complex encap... -

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Basic information

Entry
Database: EMDB / ID: EMD-1297
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 a very little substrate density (almost empty) 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: 3.793143
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.793143
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1297.gif

Downloads & links

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Map

FileDownload / File: emd_1297.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 a very little substrate density (almost empty) 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: 2.52 / Movie #1: 3.793143
Minimum - Maximum-3.02739 - 11.395200000000001
Average (Standard dev.)-0.0242212 (±1.13721)
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-3.02711.395-0.024

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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: The components for the sample were mixed with Mg-ATP at Room Temperature
Oligomeric state: GroES binds to SR398 encapsulating substrate AlphaBeta
Number unique components: 3
Molecular weightExperimental: 470 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 human mitochondrial branchedchain a-ketoacid dehydrogenase (BCKD). GroEL/GroES is essential for promoting the ...Details: Substrate AlphaBeta is an 86 kDa heterodimeric assembly intermediate of human mitochondrial branchedchain a-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.
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
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: 8280
Final angle assignmentDetails: EMAN
Final two d classificationNumber classes: 257

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