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- EMDB-12233: C. thermophilum core structure of mixed 2-oxoglutarate dehydrogen... -

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Entry
Database: EMDB / ID: EMD-12233
TitleC. thermophilum core structure of mixed 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex
Map data
Sample
  • Complex: Native core of the mixed oxoglutarate/branched-chain keto-acid dehydrogenase complex core from C. thermophilum
    • Protein or peptide: succinyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-succinyltransferase
    • Protein or peptide: 2-methylpropanoyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-(2-methylpropanoyl)transferase
Biological speciesChaetomium thermophilum var. thermophilum DSM 1495 (fungus) / Chaetomium thermophilum (fungus)
Methodsingle particle reconstruction / negative staining / Resolution: 32.7 Å
AuthorsKyrilis FL / Semchonok DA / Skalidis I / Tueting C / Hamdi F / O'Reilly FJ / Rappsilber J / Kastritis PL
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 22, 2021-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateFeb 24, 2021-
Current statusFeb 24, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0853
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0853
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12233.png

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Map

FileDownload / File: emd_12233.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 4.03 Å
Density
Contour LevelBy AUTHOR: 0.0853 / Movie #1: 0.0853
Minimum - Maximum-0.08385053 - 0.27647677
Average (Standard dev.)0.001461626 (±0.009233359)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 1007.50006 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.034.034.03
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z1007.5001007.5001007.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0840.2760.001

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Supplemental data

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Sample components

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Entire : Native core of the mixed oxoglutarate/branched-chain keto-acid de...

EntireName: Native core of the mixed oxoglutarate/branched-chain keto-acid dehydrogenase complex core from C. thermophilum
Components
  • Complex: Native core of the mixed oxoglutarate/branched-chain keto-acid dehydrogenase complex core from C. thermophilum
    • Protein or peptide: succinyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-succinyltransferase
    • Protein or peptide: 2-methylpropanoyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-(2-methylpropanoyl)transferase

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Supramolecule #1: Native core of the mixed oxoglutarate/branched-chain keto-acid de...

SupramoleculeName: Native core of the mixed oxoglutarate/branched-chain keto-acid dehydrogenase complex core from C. thermophilum
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Molecular weightTheoretical: 2 MDa

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Macromolecule #1: succinyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-succinyltransferase

MacromoleculeName: succinyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-succinyltransferase
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue succinyltransferase
Source (natural)Organism: Chaetomium thermophilum (fungus) / Strain: DSM 1495
SequenceString: MLYRGLRMAA RAAPKFAVLN THAALRQLPL QFQHVRTYAD KIIKVPQMAE SITEGTLKQW NKAVGDYVEA DEEIATIETD KIDVAVNAPE AGVIKEFFVN EEDTVLVGQD LVRLEVGGEK PAEAAKEQPK AAAPEPKVEE KKVPEAPAPE PSKTAAPAPA PPKQEAPASP ...String:
MLYRGLRMAA RAAPKFAVLN THAALRQLPL QFQHVRTYAD KIIKVPQMAE SITEGTLKQW NKAVGDYVEA DEEIATIETD KIDVAVNAPE AGVIKEFFVN EEDTVLVGQD LVRLEVGGEK PAEAAKEQPK AAAPEPKVEE KKVPEAPAPE PSKTAAPAPA PPKQEAPASP KPASKPAETP AVTLGNREER RVKMNRMRLR IAERLKQSQN TAASLTTFNE VDMSALIEFR NKYKDEVLKK TGVKLGFMSA FSRAVVLAIR DLPVVNASIE GPNGGDTIVY RDYVDISVAV ATEKGLVTPV VRNAETMDLI TIEKTIAELG KKARDGKLTI EDMAGGTFTI SNGGVFGSLM GTPIINLPQS AVLGLHAIKE RPVAVNGKVE IRPMMYLALT YDHRLLDGRE AVQFLVKVKE YIEDPRKMLL

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Macromolecule #2: 2-methylpropanoyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-(2-methyl...

MacromoleculeName: 2-methylpropanoyl-CoA:enzyme-N6-(dihydrolipoyl)lysine S-(2-methylpropanoyl)transferase
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
EC number: dihydrolipoyllysine-residue (2-methylpropanoyl)transferase
Source (natural)Organism: Chaetomium thermophilum (fungus) / Strain: DSM 1495
SequenceString: MRSWFFSRVL RQQWRVVRGL ARRAAAPTST TPLSVLSPLS AQAFHASRTL QVVKPVLLAD IGEGIVECEI IQWFVEPGAR VEEFQPLCEV QSDKASVEIT SRFAGVVKKL YYEAGEMAKV GKPFVDIDIV DGVVKEDSSA TVPIDSTPAL DKTLEAPVRP PAEGTAEAQV ...String:
MRSWFFSRVL RQQWRVVRGL ARRAAAPTST TPLSVLSPLS AQAFHASRTL QVVKPVLLAD IGEGIVECEI IQWFVEPGAR VEEFQPLCEV QSDKASVEIT SRFAGVVKKL YYEAGEMAKV GKPFVDIDIV DGVVKEDSSA TVPIDSTPAL DKTLEAPVRP PAEGTAEAQV ATATNEPTSP SKSKGKCATL ATPAVRHLSK QLGVDIAEVD GTGKDGRVLK EDIYRFVERR EAAAKQAPAT QPTASSPTPS SITSPVEGST AGSQQETPVP LTRTQEMMFK TMTRSLSIPH FLYADEVDFT KLVDLRSRLN NVLSKHGIND GQSVKLTYLP FIIKAVSMAL YQYPILNARV EIPENGNGKP MLIHRSQHNI GVAMDTPSGL LVPVIKNVGN LNILGIASEL ARLQSLAMAG KLTPQDMSGG TITVSNIGNI GGTYLSPVIV EREVAILGIG RMRTVPAFST VPGEEDKVVK KQICNFSWSA DHRVIDGATM ARAAEVVRSI VEEPDVMVMH LR

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.4 / Component - Concentration: 200.0 mM / Component - Formula: NH4CH2COOH / Component - Name: Ammonium acetate
StainingType: NEGATIVE / Material: Uranyl Acetate
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE
Detailsfractionated native cell extract

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Electron microscopy

MicroscopeTFS GLACIOS
TemperatureMin: 77.15 K / Max: 120.0 K
DetailsComa Free kept better than 200nm in EPU
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 1926 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 34739 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1407677
Details: Automated particle selection with the template derived from manual picking (selection)
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1eaa

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 32.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: The main reconstruction is octahedrally-averaged, but also all asymmetric reconstructions, half-maps and FSC curves are provided for each hybrid OGDHc/BCKDHc models are provided across studied fractions (5-11).
Number images used: 9000
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6h05

,

2ii3

)
DetailsHomology models generated for both the E2 cubic cores based on the templates 6H05 and 2II3
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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