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- EMDB-1658: Solution structure and characterisation of the human pyruvate deh... -

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Entry
Database: EMDB / ID: EMD-1658
TitleSolution structure and characterisation of the human pyruvate dehydrogenase complex core assembly
Map dataThis is a reconstruction of pyruvate dehydrogenase enzyme complex components E2 and E3BP. This contour level will display the map core, lower contour levels reveal more disordered extensions.
Sample
  • Sample: Pyruvate dehydrogenase E2-E3BP complex
  • Protein or peptide: E3 binding protein
  • Protein or peptide: Dihydrolipoyl transacetylase
KeywordsPyruvate dehydrogenase complex / dodecahedron
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.3 Å
AuthorsVijayakrishnan S / Kelly SM / Gilbert RJC / Callow P / Bhella D / Forsyth T / Lindsay JG / Byron O
CitationJournal: J Mol Biol / Year: 2010
Title: Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly.
Authors: S Vijayakrishnan / S M Kelly / R J C Gilbert / P Callow / D Bhella / T Forsyth / J G Lindsay / O Byron /
Abstract: Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a ...Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2+E3BP) core organisation: the 'addition' model (60+12) and the 'substitution' model (48+12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the 'substitution' model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.
History
DepositionOct 27, 2009-
Header (metadata) releaseOct 28, 2009-
Map releaseApr 9, 2010-
UpdateJun 1, 2010-
Current statusJun 1, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 55
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1658.gif

Downloads & links

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Map

FileDownload / File: emd_1658.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a reconstruction of pyruvate dehydrogenase enzyme complex components E2 and E3BP. This contour level will display the map core, lower contour levels reveal more disordered extensions.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.6 Å/pix.
x 128 pix.
= 460.8 Å		3.6 Å/pix.
x 128 pix.
= 460.8 Å		3.6 Å/pix.
x 128 pix.
= 460.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 55.0 / Movie #1: 55
Minimum - Maximum-56.536000000000001 - 211.342000000000013
Average (Standard dev.)5.28984 (±22.624500000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 460.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-147
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-56.536211.3425.290

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Supplemental data

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Sample components

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Entire : Pyruvate dehydrogenase E2-E3BP complex

EntireName: Pyruvate dehydrogenase E2-E3BP complex
Components
  • Sample: Pyruvate dehydrogenase E2-E3BP complex
  • Protein or peptide: E3 binding protein
  • Protein or peptide: Dihydrolipoyl transacetylase

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Supramolecule #1000: Pyruvate dehydrogenase E2-E3BP complex

SupramoleculeName: Pyruvate dehydrogenase E2-E3BP complex / type: sample / ID: 1000
Oligomeric state: A dodecahedral lattice formed of mixed trimers of E2 and E3BP, most likely each containing 2 E2 and 1 E3BP
Number unique components: 2
Molecular weightTheoretical: 4.2 MDa

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Macromolecule #1: E3 binding protein

MacromoleculeName: E3 binding protein / type: protein_or_peptide / ID: 1 / Name.synonym: E3 / Number of copies: 20 / Oligomeric state: Hetero-icosahedron / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 70 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: Dihydrolipoyl transacetylase

MacromoleculeName: Dihydrolipoyl transacetylase / type: protein_or_peptide / ID: 2 / Name.synonym: E2 / Number of copies: 40 / Oligomeric state: hetero-icosahedron / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 70 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

BufferpH: 7.5
Details: 2 mM EDTA, 0.01% (w/v) NaN3, 100 mM NaCl, 50 mM KH2PO4, pH 7.5
GridDetails: 300 mesh copper grid with lacey carbon film
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Homemade plunger
Method: Blot briefly before plunging, using Wahtman no.1 paper

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 115 K
Alignment procedureLegacy - Astigmatism: At 200,000 magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 16
Details: Images were binned from a scanned pixel size at the specimen of 1.8A to 3.6A using Proc2D
Od range: 5 / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 38000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsParticles selected interactively using Boxer
CTF correctionDetails: Per micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, WellMAP
Details: Final map had converged to given resolution, on a 1 degree spacing.
Number images used: 982
Final angle assignmentDetails: SPIDER convention

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Atomic model buiding 1

Initial modelPDB ID:

1b5s


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid body, after icosahedral symmetrization. The icosahedral assembly was constructed and then fitted as a rigid body.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CCC

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