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- EMDB-1151: Electron cryotomography of the E. coli pyruvate and 2-oxoglutarat... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-1151 | |||||||||
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Title | Electron cryotomography of the E. coli pyruvate and 2-oxoglutarate dehydrogenase complexes. | |||||||||
![]() | Volume of an extracted E. coli pyruvate dehydrogenase multienzyme complex particle from a dual-tilt tomogram. | |||||||||
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Function / homology | Pyruvate dehydrogenase E1 component / Dihydrolipoamide acetyltransferase pyruvate dehydrogenase complex / Dihydrolipoamide dehydrogenase![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 55.0 Å | |||||||||
![]() | Murphy GE / Jensen GJ | |||||||||
![]() | ![]() Title: Electron cryotomography of the E. coli pyruvate and 2-oxoglutarate dehydrogenase complexes. Authors: Gavin E Murphy / Grant J Jensen / ![]() Abstract: The E. coli pyruvate and 2-oxoglutarate dehydrogenases are two closely related, large complexes that exemplify a growing number of multiprotein "machines" whose domains have been studied extensively ...The E. coli pyruvate and 2-oxoglutarate dehydrogenases are two closely related, large complexes that exemplify a growing number of multiprotein "machines" whose domains have been studied extensively and modeled in atomic detail, but whose quaternary structures have remained unclear for lack of an effective imaging technology. Here, electron cryotomography was used to show that the E1 and E3 subunits of these complexes are flexibly tethered approximately 11 nm away from the E2 core. This result demonstrates unambiguously that electron cryotomography can reveal the relative positions of features as small as 80 kDa in individual complexes, elucidating quaternary structure and conformational flexibility. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 224.8 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.7 KB 13.7 KB | Display Display | ![]() |
Images | ![]() | 21.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 246.6 KB | Display | ![]() |
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Full document | ![]() | 245.7 KB | Display | |
Data in XML | ![]() | 4.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Volume of an extracted E. coli pyruvate dehydrogenase multienzyme complex particle from a dual-tilt tomogram. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 8.2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Unengineered E. coli Pyruvate Dehydrogenase Multienzyme Complex
Entire | Name: Unengineered E. coli Pyruvate Dehydrogenase Multienzyme Complex |
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Components |
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-Supramolecule #1000: Unengineered E. coli Pyruvate Dehydrogenase Multienzyme Complex
Supramolecule | Name: Unengineered E. coli Pyruvate Dehydrogenase Multienzyme Complex type: sample / ID: 1000 Details: The sample was thawed from storage at -80 degrees Celcius before being loaded onto the grid. Oligomeric state: Up to 24 pyruvate dehydrogenase E1p and dihydrolipoamide dehydrogenase E3 dimers together bind to the 24 dihydrolipoamide acetyltransferase E2p octahedral core. Number unique components: 3 |
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Molecular weight | Theoretical: 5.6 MDa |
-Macromolecule #1: E2p octahedral core
Macromolecule | Name: E2p octahedral core / type: protein_or_peptide / ID: 1 / Name.synonym: dihydrolipoamide acetyltransferase Details: 24 arranged as cube; See Wagenknecht, T. et al., JSB 109:70-77 (1992). Number of copies: 24 / Oligomeric state: 24mer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 1.6 MDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | InterPro: Dihydrolipoamide acetyltransferase pyruvate dehydrogenase complex |
-Macromolecule #2: E1p
Macromolecule | Name: E1p / type: protein_or_peptide / ID: 2 / Name.synonym: pyruvate dehydrogenase Details: Up to 24 of these; See Wagenknecht, T. et al., JSB 109:70-77 (1992). Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 200 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | InterPro: Pyruvate dehydrogenase E1 component |
-Macromolecule #3: E3
Macromolecule | Name: E3 / type: protein_or_peptide / ID: 3 / Name.synonym: dihydrolipoamide dehydrogenase Details: Up to 24 of these; See Wagenknecht, T. et al., JSB 109:70-77 (1992). Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 100 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | InterPro: Dihydrolipoamide dehydrogenase |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | subtomogram averaging |
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Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 7 / Details: 20 mM Potassium Phosphate |
Grid | Details: R 1.5/1.3 Quantifoil |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER Details: Vitrification instrument: Vitrobot. Sample was at 22 C in air before plunging. Method: Blot for 3.5 seconds with an offset of -3 before plunging. |
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Electron microscopy
Microscope | FEI TECNAI F30 |
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Temperature | Min: 82 K / Max: 82 K / Average: 82 K |
Alignment procedure | Legacy - Electron beam tilt params: 0 |
Specialist optics | Energy filter - Name: GIF 3000 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Date | Aug 24, 2004 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 110 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 36600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 10.0 µm / Nominal defocus min: 10.0 µm / Nominal magnification: 27500 |
Sample stage | Specimen holder: FEI Polara / Specimen holder model: GATAN HELIUM / Tilt series - Axis1 - Min angle: -63 ° / Tilt series - Axis1 - Max angle: 66 ° |
Experimental equipment | ![]() Model: Tecnai F30 / Image courtesy: FEI Company |
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Image processing
Details | Dual-axis tilt series, with 44 sections on one axis, and 45 on the other. Average number of tilts used in the 3D reconstructions: 89. Average tomographic tilt angle increment: 3. |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 55.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: ![]() Details: The individual, orthogonal tomograms were filtered at their first CTF zero and then merged. |