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- EMDB-13066: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near... -

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Basic information

Entry
Database: EMDB / ID: EMD-13066
TitleMetabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
Map dataIcosahedral symmetrized map for the E2 core of PDHc from C. thermophilum
Sample
  • Complex: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
    • 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) / Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsTueting C / Kyrilis FL / Hamdi F / Kastritis PL
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.
Authors: Christian Tüting / Fotis L Kyrilis / Johannes Müller / Marija Sorokina / Ioannis Skalidis / Farzad Hamdi / Yashar Sadian / Panagiotis L Kastritis /
Abstract: Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher- ...Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.
History
DepositionJun 10, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateDec 8, 2021-
Current statusDec 8, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ott
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ott
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13066.png

Downloads & links

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Map

FileDownload / File: emd_13066.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIcosahedral symmetrized map for the E2 core of PDHc from C. thermophilum
Voxel sizeX=Y=Z: 1.5678 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.088627614 - 0.20278731
Average (Standard dev.)-0.00013489777 (±0.0067688944)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 602.0352 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.56779947916671.56779947916671.5677994791667
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z602.035602.035602.035
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0890.203-0.000

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Supplemental data

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Mask #1

Fileemd_13066_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Asymmetric map for the E2 core of PDHc from C. thermophilum

Fileemd_13066_additional_1.map
AnnotationAsymmetric map for the E2 core of PDHc from C. thermophilum
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: C2 symmetrized map for the E2 core of PDHc from C. thermophilum

Fileemd_13066_additional_2.map
AnnotationC2 symmetrized map for the E2 core of PDHc from C. thermophilum
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Icosahedral symmetrized map for the E2 core of...

Fileemd_13066_half_map_1.map
AnnotationIcosahedral symmetrized map for the E2 core of PDHc from C. thermophilum - Half-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Icosahedral symmetrized map for the E2 core of...

Fileemd_13066_half_map_2.map
AnnotationIcosahedral symmetrized map for the E2 core of PDHc from 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 : Metabolon-embedded pyruvate dehydrogenase complex E2 core at near...

EntireName: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
Components
  • Complex: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
    • Protein or peptide: Acetyltransferase component of pyruvate dehydrogenase complex

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Supramolecule #1: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near...

SupramoleculeName: Metabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Icosahedrally symmetrized E2 core component of the pyruvate dehydrogenase complex metabolon from the thermophilic fungus Chaetomium thermophilum
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)

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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 (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719
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: C2H7NO2 / Component - Name: Ammonium ethanoate
Details: Buffer was freshly made from solid ammonium acetate, filtrated, and degassed by ultrasonication.
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Pressure: 0.04 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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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: 95677 / 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
Alignment procedureComa free - Residual tilt: 14.7 mrad
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2808 / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 296779
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: EMDB MAP
EMDB ID:

12181

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 10 / Avg.num./class: 9300 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: RANDOM ASSIGNMENT / 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: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 10249
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7bgj


Chain - Chain ID: A
DetailsThe initial model was fitted using ChimeraX and then subsequently refined using iterative cycles of manual refinement using Coot and automatic Real-space refinement using PHENIX.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7ott:
Metabolon-embedded pyruvate dehydrogenase complex E2 core at near-atomic resolution

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