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- PDB-8oiu: Cryo-EM reconstruction of the native 24-mer E2o core of the 2-oxo... -

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Basic information

Entry
Database: PDB / ID: 8oiu
TitleCryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum at 3.35 A resolution
ComponentsDihydrolipoyllysine-residue succinyltransferase
KeywordsTRANSFERASE / Dihydrolipoyl Succinyltransferase / E2 / Oxoglutarate / a-Ketoglutarate
Function / homology
Function and homology information


L-lysine catabolic process to acetyl-CoA via saccharopine / oxoglutarate dehydrogenase complex / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / tricarboxylic acid cycle
Similarity search - Function
Dihydrolipoamide succinyltransferase / 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 succinyltransferase
Similarity search - Component
Biological speciesThermochaetoides thermophila DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsSkalidis, I. / Tueting, C. / Kyrilis, F.L. / Hamdi, F. / Kastritis, P.L.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Commun Biol / Year: 2023
Title: Structural analysis of an endogenous 4-megadalton succinyl-CoA-generating metabolon.
Authors: Ioannis Skalidis / Fotis L Kyrilis / Christian Tüting / Farzad Hamdi / Toni K Träger / Jaydeep Belapure / Gerd Hause / Marta Fratini / Francis J O'Reilly / Ingo Heilmann / Juri Rappsilber ...Authors: Ioannis Skalidis / Fotis L Kyrilis / Christian Tüting / Farzad Hamdi / Toni K Träger / Jaydeep Belapure / Gerd Hause / Marta Fratini / Francis J O'Reilly / Ingo Heilmann / Juri Rappsilber / Panagiotis L Kastritis /
Abstract: The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. ...The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. Due to its pivotal role in metabolism, OGDHc enzymatic components have been studied in isolation; however, their interactions within the endogenous OGDHc remain elusive. Here, we discern the organization of a thermophilic, eukaryotic, native OGDHc in its active state. By combining biochemical, biophysical, and bioinformatic methods, we resolve its composition, 3D architecture, and molecular function at 3.35 Å resolution. We further report the high-resolution cryo-EM structure of the OGDHc core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of OGDHc participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-subunit communication, and the presence of a flexible subunit (E3BPo), connecting E2o and E3. This multi-scale analysis of a succinyl-CoA-producing native cell extract provides a blueprint for structure-function studies of complex mixtures of medical and biotechnological value.
History
DepositionMar 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 31, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dihydrolipoyllysine-residue succinyltransferase


Theoretical massNumber of molelcules
Total (without water)45,9871
Polymers45,9871
Non-polymers00
Water0
1
A: Dihydrolipoyllysine-residue succinyltransferase
x 24


Theoretical massNumber of molelcules
Total (without water)1,103,68824
Polymers1,103,68824
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation23

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Components

#1: Protein Dihydrolipoyllysine-residue succinyltransferase /


Mass: 45987.004 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Thermochaetoides thermophila DSM 1495 (fungus)
References: UniProt: G0SAX9, dihydrolipoyllysine-residue succinyltransferase

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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 24-mer core of Oxoglutarate Dehydrogenase Complex
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 3 MDa / Experimental value: NO
Source (natural)Organism: Thermochaetoides thermophila 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
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: For plunging, blot force 0 and blotting time of 4 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: 92000 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
Image recordingElectron dose: 30 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 21381

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.1particle selection
2EPU2.6image acquisitionData collection
4cryoSPARC3.1CTF correctionPatch CTF
7UCSF ChimeraX1.2model fittingRigid fitting
9PHENIX1.19model refinementReal-space refinement
10cryoSPARC3.1initial Euler assignmentAb-initio reconstruction
11cryoSPARC3.1final Euler assignmentLocal refinement (NEW)
12cryoSPARC3.1classification2D classification
13cryoSPARC3.13D reconstructionLocal refinement (NEW)
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1300 / Details: Template picking was directly applied
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69028 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT)
Details: Particles were symmetry expanded for the final reconstruction, then local reconstruction was performed without symmetry application.
Num. of class averages: 50 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial fitting was performed in ChimeraX v1.2, then real-space refined in PHENIX v1.19
Atomic model buildingPDB-ID: 7Q5Q

7q5q


Pdb chain-ID: all / Accession code: 7Q5Q / Source name: PDB / Type: experimental model

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