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- EMDB-12234: C. thermophilum Pyruvate Dehydrogenase Complex Core from native c... -

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Basic information

Entry
Database: EMDB / ID: EMD-12234
TitleC. thermophilum Pyruvate Dehydrogenase Complex Core from native cell extracts
Map data
Sample
  • Complex: Native core of the Pyruvate Dehydrogenase Complex from C. thermophilum
    • Protein or peptide: Dihydrolipoyl transacylase, Pyruvate Dehydrogenase Complex E2 component
Biological speciesChaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Methodsingle particle reconstruction / negative staining / Resolution: 14.2 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: 0.0747
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0747
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12234.png

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Map

FileDownload / File: emd_12234.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
4.03 Å/pix.
x 250 pix.
= 1007.5 Å		4.03 Å/pix.
x 250 pix.
= 1007.5 Å		4.03 Å/pix.
x 250 pix.
= 1007.5 Å

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Voxel sizeX=Y=Z: 4.03 Å
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Contour LevelBy AUTHOR: 0.0747 / Movie #1: 0.0747
Minimum - Maximum-0.051921524 - 0.17748983
Average (Standard dev.)-0.00049081567 (±0.010953227)
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-0.0520.177-0.000

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Supplemental data

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Additional map: #18

Fileemd_12234_additional_1.map
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Sample components

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Entire : Native core of the Pyruvate Dehydrogenase Complex from C. thermophilum

EntireName: Native core of the Pyruvate Dehydrogenase Complex from C. thermophilum
Components
  • Complex: Native core of the Pyruvate Dehydrogenase Complex from C. thermophilum
    • Protein or peptide: Dihydrolipoyl transacylase, Pyruvate Dehydrogenase Complex E2 component

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Supramolecule #1: Native core of the Pyruvate Dehydrogenase Complex from C. thermophilum

SupramoleculeName: Native core of the Pyruvate Dehydrogenase Complex from C. thermophilum
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Molecular weightTheoretical: 3 MDa

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Macromolecule #1: Dihydrolipoyl transacylase, Pyruvate Dehydrogenase Complex E2 com...

MacromoleculeName: Dihydrolipoyl transacylase, Pyruvate Dehydrogenase Complex E2 component
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue acetyltransferase
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
SequenceString: MLAQVLRRQA LQHVRLARAA APSLTRWYAS YPPHTIVKMP ALSPTMTSGN IGAWQKKPGD AITPGEVLVE IETDKAQMDF EFQEEGVLAK ILKETGEKDV AVGSPIAVLV EEGTDINAFQ NFTLEDAGGD AAAPAAPAKE ELAKAETAPT PASTSAPEPE ETTSTGKLEP ...String:
MLAQVLRRQA LQHVRLARAA APSLTRWYAS YPPHTIVKMP ALSPTMTSGN IGAWQKKPGD AITPGEVLVE IETDKAQMDF EFQEEGVLAK ILKETGEKDV AVGSPIAVLV EEGTDINAFQ NFTLEDAGGD AAAPAAPAKE ELAKAETAPT PASTSAPEPE ETTSTGKLEP ALDREPNVSF AAKKLAHELD VPLKALKGTG PGGKITEEDV KKAASAPAAA AAAPGAAYQD IPISNMRKTI ATRLKESVSE NPHFFVTSEL SVSKLLKLRQ ALNSSAEGRY KLSVNDFLIK AIAVACKRVP AVNSSWRDGV IRQFDTVDVS VAVATPTGLI TPIVKGVEAK GLETISATVK ELAKKARDGK LKPEDYQGGT ISISNMGMNP AVERFTAIIN PPQAAILAVG TTKKVAVPVE NEDGTTGVEW DDQIVVTASF DHKVVDGAVG AEWMRELKKV VENPLELLL

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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:

6ct0

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 14.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 267547
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 modelPDB ID:

6ct0


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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