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- EMDB-16900: Cryo-EM reconstruction of the native 24-mer E2o core of the 2-oxo... -

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Basic information

Entry
Database: EMDB / ID: EMD-16900
TitleCryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum at 3.35 A resolution
Map dataCryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum.
Sample
  • Complex: Native 24-mer core of Oxoglutarate Dehydrogenase Complex
    • Protein or peptide: Dihydrolipoyllysine-residue succinyltransferase
KeywordsDihydrolipoyl Succinyltransferase / E2 / Oxoglutarate / a-Ketoglutarate / TRANSFERASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsSkalidis I / Tueting C / Kyrilis FL / Hamdi F / Kastritis PL
Funding support1 items
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, 2023-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16900.png

Downloads & links

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Map

FileDownload / File: emd_16900.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.57 Å/pix.
x 208 pix.
= 326.102 Å		1.57 Å/pix.
x 208 pix.
= 326.102 Å		1.57 Å/pix.
x 208 pix.
= 326.102 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5678 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-2.3572903 - 5.9476595
Average (Standard dev.)-0.003834117 (±0.22103816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 326.10242 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16900_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-EM reconstruction of the native asymmetric complex of...

Fileemd_16900_additional_1.map
AnnotationCryo-EM reconstruction of the native asymmetric complex of the 2-oxoglutarate dehydrogenase complex of C. thermophilum.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM reconstruction of the native 24-mer E2o core...

Fileemd_16900_half_map_1.map
AnnotationCryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum, Half-Map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM reconstruction of the native 24-mer E2o core...

Fileemd_16900_half_map_2.map
AnnotationCryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum, Half-Map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Native 24-mer core of Oxoglutarate Dehydrogenase Complex

EntireName: Native 24-mer core of Oxoglutarate Dehydrogenase Complex
Components
  • Complex: Native 24-mer core of Oxoglutarate Dehydrogenase Complex
    • Protein or peptide: Dihydrolipoyllysine-residue succinyltransferase

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Supramolecule #1: Native 24-mer core of Oxoglutarate Dehydrogenase Complex

SupramoleculeName: Native 24-mer core of Oxoglutarate Dehydrogenase Complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 3 MDa

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Macromolecule #1: Dihydrolipoyllysine-residue succinyltransferase

MacromoleculeName: Dihydrolipoyllysine-residue succinyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue succinyltransferase
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 45.987004 KDa
SequenceString: MLYRGLRMAA RAAPKFAVLN THAALRQLPL QFQHVRTYAD KIIKVPQMAE SITEGTLKQW NKAVGDYVEA DEEIATIETD KIDVAVNAP EAGVIKEFFV NEEDTVLVGQ DLVRLEVGGE KPAEAAKEQP KAAAPEPKVE EKKVPEAPAP EPSKTAAPAP A PPKQEAPA ...String:
MLYRGLRMAA RAAPKFAVLN THAALRQLPL QFQHVRTYAD KIIKVPQMAE SITEGTLKQW NKAVGDYVEA DEEIATIETD KIDVAVNAP EAGVIKEFFV NEEDTVLVGQ DLVRLEVGGE KPAEAAKEQP KAAAPEPKVE EKKVPEAPAP EPSKTAAPAP A PPKQEAPA SPKPASKPAE TPAVTLGNRE ERRVKMNRMR LRIAERLKQS QNTAASLTTF NEVDMSALIE FRNKYKDEVL KK TGVKLGF MSAFSRAVVL AIRDLPVVNA SIEGPNGGDT IVYRDYVDIS VAVATEKGLV TPVVRNAETM DLITIEKTIA ELG KKARDG KLTIEDMAGG TFTISNGGVF GSLMGTPIIN LPQSAVLGLH AIKERPVAVN GKVEIRPMMY LALTYDHRLL DGRE AVQFL VKVKEYIEDP RKMLL

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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

Concentration0.3 mg/mL
BufferpH: 7.4 / Component - Concentration: 200.0 mM / Component - Formula: NH4CH2COOH / Component - Name: Ammonium acetate
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: For plunging, blot force 0 and blotting time of 4 sec were applied..

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.15 K / Max: 103.15 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number real images: 21381 / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 1300 / Details: Template picking was directly applied
Startup modelType of model: EMDB MAP
EMDB ID:

13844

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1) / Software - details: Ab-initio reconstruction
Final 3D classificationNumber classes: 200 / Avg.num./class: 2133 / Software - Name: cryoSPARC (ver. 3.1) / Software - details: 2D classification
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) / Software - details: Local refinement (NEW)
Final reconstructionNumber classes used: 50 / Applied symmetry - Point group: O (octahedral) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Software - details: Local refinement (NEW)
Details: Particles were symmetry expanded for the final reconstruction, then local reconstruction was performed without symmetry application.
Number images used: 69028
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial fitting was performed in ChimeraX v1.2, then real-space refined in PHENIX v1.19
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8oiu:
Cryo-EM reconstruction of the native 24-mer E2o core of the 2-oxoglutarate dehydrogenase complex of C. thermophilum at 3.35 A resolution

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