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- PDB-7ott: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near... -

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Entry
Database: PDB / ID: 7ott
TitleMetabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
ComponentsAcetyltransferase component of pyruvate dehydrogenase complex
KeywordsTRANSFERASE / pyruvate / dehydrogenase / complex / e2 / core / c.thermophilum / metabolon
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 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsTueting, C. / Kastritis, P.L.
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.
Authors: Christian Tüting / Fotis L Kyrilis / Johannes Müller / Marija Sorokina / Ioannis Skalidis / Farzad Hamdi / Yashar Sadian / Panagiotis L Kastritis /
Abstract: Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher- ...Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.
History
DepositionJun 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 8, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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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: microscopy, In our 3D reconstruction, we applied icosahedral symmetry to reach a high resolution. The native assembly is therefore an icosahedral complex, consisting of 60 identical ...Evidence: microscopy, In our 3D reconstruction, we applied icosahedral symmetry to reach a high resolution. The native assembly is therefore an icosahedral complex, consisting of 60 identical polypeptide chains., gel filtration, The native protein complex elutes at a very high molecular weight (>5 MDa), indicating a fully assembled metabolon., homology, The core structure is an icosahedral structure, also seen in the structures 6ZLM, 6CT0, and 7BGJ.
  • 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 (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719
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: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
Type: COMPLEX
Details: Icosahedrally symmetrized E2 core component of the pyruvate dehydrogenase complex metabolon from the thermophilic fungus Chaetomium thermophilum
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Buffer solutionpH: 7.4
Details: Buffer was freshly made from solid ammonium acetate, filtrated, and degassed by ultrasonication.
Buffer componentConc.: 200 mM / Name: Ammonium ethanoate / Formula: C2H7NO2
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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: 92000 X / Calibrated magnification: 95677 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 grids imaged: 1 / Num. of real images: 2808
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18_3845refinement
PHENIX1.18_3845refinement
EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2EPU2.9image acquisition
4Gctf1.06CTF correction
7UCSF ChimeraX1.2model fitting
9PHENIX1.18-3845model refinementReal-space Refinement
10Coot0.9.4model refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 296779
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10249 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: The initial model was fitted using ChimeraX and then subsequently refined using iterative cycles of manual refinement using Coot and automatic Real-space refinement using PHENIX.
Atomic model buildingPDB-ID: 7BGJ

7bgj


Pdb chain-ID: A
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 63.92 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311608
ELECTRON MICROSCOPYf_angle_d0.72142185
ELECTRON MICROSCOPYf_chiral_restr0.046265
ELECTRON MICROSCOPYf_plane_restr0.0045279
ELECTRON MICROSCOPYf_dihedral_angle_d15.6159594

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