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- EMDB-13845: Protein community member pyruvate dehydrogenase complex E2 core f... -

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Basic information

Entry
Database: EMDB / ID: EMD-13845
TitleProtein community member pyruvate dehydrogenase complex E2 core from C. thermophilum
Map dataC. thermophilum Native Community Member Pyruvate Dehydrogenase Complex E2 Core
Sample
  • Complex: Native 60-mer core of Pyruvate Dehydrogenase Complex
    • Protein or peptide: Acetyltransferase component of pyruvate dehydrogenase complex
KeywordsDihydrolipoyl / transacetylase / E2 / Pyruvate / TRANSFERASE
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
AuthorsSkalidis I / Kyrilis FL / Tueting C / Hamdi F / Kastritis PL
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Structure / Year: 2022
Title: Cryo-EM and artificial intelligence visualize endogenous protein community members.
Authors: Ioannis Skalidis / Fotis L Kyrilis / Christian Tüting / Farzad Hamdi / Grzegorz Chojnowski / Panagiotis L Kastritis /
Abstract: Cellular function is underlined by megadalton assemblies organizing in proximity, forming communities. Metabolons are protein communities involving metabolic pathways such as protein, fatty acid, and ...Cellular function is underlined by megadalton assemblies organizing in proximity, forming communities. Metabolons are protein communities involving metabolic pathways such as protein, fatty acid, and thioesters of coenzyme-A synthesis. Metabolons are highly heterogeneous due to their function, making their analysis particularly challenging. Here, we simultaneously characterize metabolon-embedded architectures of a 60S pre-ribosome, fatty acid synthase, and pyruvate/oxoglutarate dehydrogenase complex E2 cores de novo. Cryo-electron microscopy (cryo-EM) 3D reconstructions are resolved at 3.84-4.52 Å resolution by collecting <3,000 micrographs of a single cellular fraction. After combining cryo-EM with artificial intelligence-based atomic modeling and de novo sequence identification methods, at this resolution range, polypeptide hydrogen bonding patterns are discernible. Residing molecular components resemble their purified counterparts from other eukaryotes but also exhibit substantial conformational variation with potential functional implications. Our results propose an integrated tool, boosted by machine learning, that opens doors for structural systems biology spearheaded by cryo-EM characterization of native cell extracts.
History
DepositionNov 4, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7q5r
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7q5r
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13845.png

Downloads & links

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Map

FileDownload / File: emd_13845.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC. thermophilum Native Community Member Pyruvate Dehydrogenase Complex E2 Core
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.57 Å/pix.
x 384 pix.
= 602.035 Å		1.57 Å/pix.
x 384 pix.
= 602.035 Å		1.57 Å/pix.
x 384 pix.
= 602.035 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: 0.9 / Movie #1: 0.9
Minimum - Maximum-1.4152321 - 3.9216099
Average (Standard dev.)0.0029887024 (±0.14969647)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-1.4153.9220.003

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Supplemental data

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Mask #1

Fileemd_13845_msk_1.map
Projections & Slices
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Additional map: C. thermophilum Native Community Member Signature 2 Ab Initio Model

Fileemd_13845_additional_1.map
AnnotationC. thermophilum Native Community Member Signature 2 Ab Initio Model
Projections & Slices
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Half map: C. thermophilum Native Community Member Pyruvate Dehydrogenase Complex...

Fileemd_13845_half_map_1.map
AnnotationC. thermophilum Native Community Member Pyruvate Dehydrogenase Complex E2 Core, Half-Map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: C. thermophilum Native Community Member Pyruvate Dehydrogenase Complex...

Fileemd_13845_half_map_2.map
AnnotationC. thermophilum Native Community Member Pyruvate Dehydrogenase Complex E2 Core, Half-Map A
Projections & Slices
AxesZYX

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Slices (1/2)
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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)

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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: 60 / 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

UniProtKB: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial

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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 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
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: 2808 / Average electron dose: 30.0 e/Å2

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Image processing

Particle selectionNumber selected: 276399
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7bgj

Initial angle assignmentType: RANDOM ASSIGNMENT
Final 3D classificationNumber classes: 256 / Avg.num./class: 1000 / Software - Name: cryoSPARC (ver. 3.1) / Software - details: 2D Classification
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied 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: cryoSPARC (ver. 3.1) / Software - details: Homogeneous Refinement (NEW) / Number images used: 7825
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial models were predicted using AlphaFold v2.0.1, fitted into reconstructions using COOT and finally refined in real space using COOT and phenix.real_space_refine
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7q5r:
Protein community member pyruvate dehydrogenase complex E2 core from C. thermophilum

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