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- EMDB-12181: Cryo-EM map of Icosahedrally averaged native core of Pyruvate Deh... -

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Basic information

Entry
Database: EMDB / ID: EMD-12181
TitleCryo-EM map of Icosahedrally averaged native core of Pyruvate Dehydrogenase Complex from Ch. thermophilum
Map dataSymmetrically averaged (Icosahedral Symmetry) Pyruvate Dehydrogenase Complex core from Ch. thermophilum.
Sample
  • Complex: Native 60-mer core of Pyruvate Dehydrogenase Complex
    • Protein or peptide: Acetyltransferase component of pyruvate dehydrogenase complex
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 var. thermophilum DSM 1495 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsSkalidis I / Kyrilis FL / Tueting C / Semchonok DA / 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 7, 2021-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateFeb 10, 2021-
Current statusFeb 10, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bgj
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bgj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12181.png

Downloads & links

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Map

FileDownload / File: emd_12181.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetrically averaged (Icosahedral Symmetry) Pyruvate Dehydrogenase Complex core from Ch. thermophilum.
Voxel sizeX=Y=Z: 3.1765 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.026
Minimum - Maximum-0.057240035 - 0.12619737
Average (Standard dev.)0.00019970845 (±0.0066095362)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 794.125 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.17653.17653.1765
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z794.125794.125794.125
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0570.1260.000

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Supplemental data

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Additional map: Asymmetrical Pyruvate Dehydrogenase Complex from Ch. thermophilum.

Fileemd_12181_additional_1.map
AnnotationAsymmetrical Pyruvate Dehydrogenase Complex from Ch. thermophilum.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Asymmetrical Pyruvate Dehydrogenase Complex from Ch. thermophilum, Half-map...

Fileemd_12181_additional_2.map
AnnotationAsymmetrical Pyruvate Dehydrogenase Complex from Ch. thermophilum, Half-map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Asymmetrical Pyruvate Dehydrogenase Complex from Ch. thermophilum, Half-map...

Fileemd_12181_additional_3.map
AnnotationAsymmetrical Pyruvate Dehydrogenase Complex from Ch. thermophilum, Half-map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Asymmetrical Pyruvate Dehydrogenase Complex core from Ch. thermophilum....

Fileemd_12181_additional_4.map
AnnotationAsymmetrical Pyruvate Dehydrogenase Complex core from Ch. thermophilum. Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Asymmetrical Pyruvate Dehydrogenase Complex core from Ch. thermophilum....

Fileemd_12181_additional_5.map
AnnotationAsymmetrical Pyruvate Dehydrogenase Complex core from Ch. thermophilum. Half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Asymmetrical Pyruvate Dehydrogenase Complex core from Ch. thermophilum....

Fileemd_12181_additional_6.map
AnnotationAsymmetrical Pyruvate Dehydrogenase Complex core from Ch. thermophilum.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Symmetrically averaged (Icosahedral Symmetry) Pyruvate Dehydrogenase Complex core...

Fileemd_12181_half_map_1.map
AnnotationSymmetrically averaged (Icosahedral Symmetry) Pyruvate Dehydrogenase Complex core from Ch. thermophilum, Half-map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Symmetrically averaged (Icosahedral Symmetry) Pyruvate Dehydrogenase Complex core...

Fileemd_12181_half_map_2.map
AnnotationSymmetrically averaged (Icosahedral Symmetry) Pyruvate Dehydrogenase Complex core from Ch. thermophilum, Half-map 2.
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 60-mer core of Pyruvate Dehydrogenase Complex

EntireName: Native 60-mer core of Pyruvate Dehydrogenase Complex
Components
  • Complex: Native 60-mer core of Pyruvate Dehydrogenase Complex
    • Protein or peptide: Acetyltransferase component of pyruvate dehydrogenase complex

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Supramolecule #1: Native 60-mer core of Pyruvate Dehydrogenase Complex

SupramoleculeName: Native 60-mer core of Pyruvate Dehydrogenase Complex / 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: Acetyltransferase component of pyruvate dehydrogenase complex

MacromoleculeName: Acetyltransferase component of pyruvate dehydrogenase complex
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue acetyltransferase
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Molecular weightTheoretical: 48.777488 KDa
SequenceString: MLAQVLRRQA LQHVRLARAA APSLTRWYAS YPPHTIVKMP ALSPTMTSGN IGAWQKKPGD AITPGEVLVE IETDKAQMDF EFQEEGVLA KILKETGEKD VAVGSPIAVL VEEGTDINAF QNFTLEDAGG DAAAPAAPAK EELAKAETAP TPASTSAPEP E ETTSTGKL ...String:
MLAQVLRRQA LQHVRLARAA APSLTRWYAS YPPHTIVKMP ALSPTMTSGN IGAWQKKPGD AITPGEVLVE IETDKAQMDF EFQEEGVLA KILKETGEKD VAVGSPIAVL VEEGTDINAF QNFTLEDAGG DAAAPAAPAK EELAKAETAP TPASTSAPEP E ETTSTGKL EPALDREPNV SFAAKKLAHE LDVPLKALKG TGPGGKITEE DVKKAASAPA AAAAAPGAAY QDIPISNMRK TI ATRLKES VSENPHFFVT SELSVSKLLK LRQALNSSAE GRYKLSVNDF LIKAIAVACK RVPAVNSSWR DGVIRQFDTV DVS VAVATP TGLITPIVKG VEAKGLETIS ATVKELAKKA RDGKLKPEDY QGGTISISNM GMNPAVERFT AIINPPQAAI LAVG TTKKV AVPVENEDGT TGVEWDDQIV VTASFDHKVV DGAVGAEWMR ELKKVVENPL ELLL

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: For plunging, blot force 2 and blotting time of 6 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 / Calibrated magnification: 44067 / 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: 45000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.15 K / Max: 103.15 K
Alignment procedureComa free - Residual tilt: 14.7 mrad
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number real images: 2593 / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 205251
Details: Automated selection with template derived from manual selection
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ct0

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 29516
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ct0


Chain - Chain ID: A / Chain - Residue range: 444-647
DetailsThe initial model was used to generate our model using MODELLER. This inital model was fitted into the density using ChimeraX and refined in real space using PHENIX.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 103.25
Output model

PDB-7bgj:
C. thermophilum Pyruvate Dehydrogenase Complex Core

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