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- PDB-7bgj: C. thermophilum Pyruvate Dehydrogenase Complex Core -

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Basic information

Entry
Database: PDB / ID: 7bgj
TitleC. thermophilum Pyruvate Dehydrogenase Complex Core
ComponentsAcetyltransferase component of pyruvate dehydrogenase complex
KeywordsTRANSFERASE / Dihydrolipoyl / transacetylase / E2 / Pyruvate
Function / homology
Function and homology information


dihydrolipoyllysine-residue acetyltransferase / dihydrolipoyllysine-residue acetyltransferase activity / acetyl-CoA biosynthetic process from pyruvate / pyruvate dehydrogenase complex / mitochondrion
Similarity search - Function
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex / Dihydrolipoamide acetyltransferase/Pyruvate dehydrogenase protein X component / Peripheral subunit-binding domain / e3 binding domain / Peripheral subunit-binding (PSBD) domain profile. / E3-binding domain superfamily / 2-oxo acid dehydrogenase, lipoyl-binding site / 2-oxo acid dehydrogenases acyltransferase component lipoyl binding site. / 2-oxoacid dehydrogenase acyltransferase, catalytic domain / 2-oxoacid dehydrogenases acyltransferase (catalytic domain) ...Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex / Dihydrolipoamide acetyltransferase/Pyruvate dehydrogenase protein X component / Peripheral subunit-binding domain / e3 binding domain / Peripheral subunit-binding (PSBD) domain profile. / E3-binding domain superfamily / 2-oxo acid dehydrogenase, lipoyl-binding site / 2-oxo acid dehydrogenases acyltransferase component lipoyl binding site. / 2-oxoacid dehydrogenase acyltransferase, catalytic domain / 2-oxoacid dehydrogenases acyltransferase (catalytic domain) / Biotin-requiring enzyme / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / Single hybrid motif / Chloramphenicol acetyltransferase-like domain superfamily
Similarity search - Domain/homology
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial
Similarity search - Component
Biological speciesChaetomium thermophilum var. thermophilum DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsTueting, C. / Kastritis, P.L.
CitationJournal: Cell Rep / Year: 2021
Title: Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts.
Authors: Fotis L Kyrilis / Dmitry A Semchonok / Ioannis Skalidis / Christian Tüting / Farzad Hamdi / Francis J O'Reilly / Juri Rappsilber / Panagiotis L Kastritis /
Abstract: The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure ...The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.
History
DepositionJan 7, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Acetyltransferase component of pyruvate dehydrogenase complex


Theoretical massNumber of molelcules
Total (without water)48,7771
Polymers48,7771
Non-polymers00
Water0
1
A: Acetyltransferase component of pyruvate dehydrogenase complex
x 60


  • defined by author
  • Evidence: gel filtration, We isolated the protein complex by using only the heavy fractions of a Phenomenex Biosep SEC-s4000 with an upper exlcusion limit in the MDa-range., scanning transmission ...Evidence: gel filtration, We isolated the protein complex by using only the heavy fractions of a Phenomenex Biosep SEC-s4000 with an upper exlcusion limit in the MDa-range., scanning transmission electron microscopy, We performed Cryo-TEM on a vitrified, fractionated cell extract, where, after systematic image analysis we ended up with an Icosahedral core for the Pyruvate Dehydrogenase Complex., homology, It is known that the E2 protein of the Pyruvate Dehydrogenase Complex forms an icosahedral assembly, e.g. in the human counterpart (PDB-ID: 6ct0)
  • 2.93 MDa, 60 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)2,926,64960
Polymers2,926,64960
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation59

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Components

#1: Protein Acetyltransferase component of pyruvate dehydrogenase complex /


Mass: 48777.488 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
References: UniProt: G0S4X6, dihydrolipoyllysine-residue acetyltransferase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Native 60-mer core of Pyruvate Dehydrogenase Complex / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 3 MDa / Experimental value: NO
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Buffer solutionpH: 7.4
Buffer componentConc.: 200 mM / Name: Ammonium acetate / Formula: NH4CH2COOH
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: For plunging, blot force 2 and blotting time of 6 sec were applied.

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Calibrated magnification: 44067 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 103.15 K / Temperature (min): 77.15 K / Residual tilt: 14.7 mradians
Image recordingElectron dose: 30 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 2593

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3845refinement
PHENIX1.18_3845refinement
EM software
IDNameVersionCategoryDetails
1Xmipp3particle selection
2EPU2.6image acquisitionImage Collection
3Scipion2image acquisitionImage analysis software integration
4RELION3image acquisitionParticle extraction, 2D/3D classification, Model refinement
5Xmipp3image acquisitionParticle picking
6MotionCorr22.03image acquisitionMotion correction
7Gctf1.06image acquisitionCTF estimation
9Gctf1.06CTF correction
12UCSF Chimera0.94model fittingUCSF ChimeraX
14RELION3initial Euler assignment
15RELION3final Euler assignment
16RELION3classification
17RELION33D reconstruction
24MODELLER9.22model refinement
25PHENIX1.18-3845model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 205251
Details: Automated selection with template derived from manual selection
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29516 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 103.25 / Protocol: FLEXIBLE FIT / Space: REAL
Details: The initial model was used to generate our model using MODELLER. This inital model was fitted into the density using ChimeraX and refined in real space using PHENIX.
Atomic model buildingPDB-ID: 6CT0

6ct0


Pdb chain-ID: A / Pdb chain residue range: 444-647
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 724.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00751600
ELECTRON MICROSCOPYf_angle_d1.11342173
ELECTRON MICROSCOPYf_chiral_restr0.0595264
ELECTRON MICROSCOPYf_plane_restr0.0061276
ELECTRON MICROSCOPYf_dihedral_angle_d22.7131591

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