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- EMDB-0108: Cryo-electron microscopic structure of the dihydrolipoamide succi... -

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Basic information

Entry
Database: EMDB / ID: EMD-0108
TitleCryo-electron microscopic structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate (2-oxoglutarate) dehydrogenase complex [residues 218-453]
Map dataCryo-electron microscopic map of the human dihydrolipoamide succinyltransferase [residues 218-453]
Sample
  • Complex: Affinity purified human dihydrolipoamide succinyltransferase
    • Protein or peptide: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial
Function / homology
Function and homology information


succinyl-CoA metabolic process / : / L-lysine catabolic process to acetyl-CoA via saccharopine / oxoglutarate dehydrogenase complex / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / Lysine catabolism / Citric acid cycle (TCA cycle) / 2-oxoglutarate metabolic process / Glyoxylate metabolism and glycine degradation ...succinyl-CoA metabolic process / : / L-lysine catabolic process to acetyl-CoA via saccharopine / oxoglutarate dehydrogenase complex / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / Lysine catabolism / Citric acid cycle (TCA cycle) / 2-oxoglutarate metabolic process / Glyoxylate metabolism and glycine degradation / acyltransferase activity / centriolar satellite / tricarboxylic acid cycle / generation of precursor metabolites and energy / mitochondrial matrix / mitochondrion / nucleoplasm / membrane / nucleus / cytosol
Similarity search - Function
Dihydrolipoamide succinyltransferase / 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 succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsNagy B / Zambo Z / Hubert A / Polak M / Nemeria NS / Novacek J / Jordan F / Adam-Vizi V / Ambrus A
Funding support Hungary, United States, 6 items
OrganizationGrant numberCountry
Hungarian Academy of SciencesMTA grant 02001 (to Vera Adam-Vizi) Hungary
Hungarian Academy of SciencesOTKA grant 112230 (to Vera Adam-Vizi) Hungary
Hungarian Academy of SciencesNAP grant KTIA_13_NAP-A-III/6 (to Vera Adam-Vizi) Hungary
Hungarian Academy of SciencesNAP 2 grant 2017-1.2.1-NKP- 2017-00002 (to Vera Adam-Vizi) Hungary
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH-GM-050380 (to Frank Jordan) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R15-GM116077 (to Frank Jordan) United States
CitationJournal: Biochim Biophys Acta Gen Subj / Year: 2021
Title: Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: ...Title: Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure.
Authors: Balint Nagy / Martin Polak / Oliver Ozohanics / Zsofia Zambo / Eszter Szabo / Agnes Hubert / Frank Jordan / Jiří Novaček / Vera Adam-Vizi / Attila Ambrus /
Abstract: BACKGROUND: The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as ...BACKGROUND: The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers.
METHODS: We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl ...METHODS: We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling.
RESULTS: We report the 2.9 Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few ...RESULTS: We report the 2.9 Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1-150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc.
CONCLUSIONS: The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in ...CONCLUSIONS: The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly.
GENERAL SIGNIFICANCE: The revealed structural information will support the future pharmacologically targeting of the hKGDHc.
History
DepositionJul 6, 2018-
Header (metadata) releaseJan 22, 2020-
Map releaseJan 22, 2020-
UpdateApr 7, 2021-
Current statusApr 7, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6h05
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6h05
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0108.png

Downloads & links

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Map

FileDownload / File: emd_0108.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron microscopic map of the human dihydrolipoamide succinyltransferase [residues 218-453]
Voxel sizeX=Y=Z: 1.061 Å
Density
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.8
Minimum - Maximum-2.941883 - 5.5731964
Average (Standard dev.)0.0015354434 (±0.18934834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-140-140-140
Dimensions280280280
Spacing280280280
CellA=B=C: 297.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0611.0611.061
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z297.080297.080297.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-140-140-140
NC/NR/NS280280280
D min/max/mean-2.9425.5730.002

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Supplemental data

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Sample components

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Entire : Affinity purified human dihydrolipoamide succinyltransferase

EntireName: Affinity purified human dihydrolipoamide succinyltransferase
Components
  • Complex: Affinity purified human dihydrolipoamide succinyltransferase
    • Protein or peptide: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial

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Supramolecule #1: Affinity purified human dihydrolipoamide succinyltransferase

SupramoleculeName: Affinity purified human dihydrolipoamide succinyltransferase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Protein in 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET52b+

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Macromolecule #1: Dihydrolipoyllysine-residue succinyltransferase component of 2-ox...

MacromoleculeName: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial
type: protein_or_peptide / ID: 1
Details: N-terminal Twin-Strep (affinity) tag with proteolytic cleavage site and linkers: MASWSHPQFEKGGGSGGGSGGSAWSHPQFEKLEVLFQGPG Density was found for 236 residues [218-453]
Number of copies: 1 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue succinyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.543004 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KLEVLFQGPG DDLVTVKTPA FAESVTEGDV RWEKAVGDTV AEDEVVCEIE TDKTSVQVP SPANGVIEAL LVPDGGKVEG GTPLFTLRKT GAAPAKAKPA EAPAAAAPKA EPTAAAVPPP AAPIPTQMPP V PSPSQPPS ...String:
MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KLEVLFQGPG DDLVTVKTPA FAESVTEGDV RWEKAVGDTV AEDEVVCEIE TDKTSVQVP SPANGVIEAL LVPDGGKVEG GTPLFTLRKT GAAPAKAKPA EAPAAAAPKA EPTAAAVPPP AAPIPTQMPP V PSPSQPPS GKPVSAVKPT VAPPLAEPGA GKGLRSEHRE KMNRMRQRIA QRLKEAQNTC AMLTTFNEID MSNIQEMRAR HK EAFLKKH NLKLGFMSAF VKASAFALQE QPVVNAVIDD TTKEVVYRDY IDISVAVATP RGLVVPVIRN VEAMNFADIE RTI TELGEK ARKNELAIED MDGGTFTISN GGVFGSLFGT PIINPPQSAI LGMHGIFDRP VAIGGKVEVR PMMYVALTYD HRLI DGREA VTFLRKIKAA VEDPRVLLLD L

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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

Concentration1.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNH2C(CH2OH)3HClTris-HClTris
150.0 mMNaClSodium chlorideSodium cloride
1.0 mMC10H16N2O8Ethylenediaminetetraacetic acid
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was highly purified and monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 48.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1533026
Details: Manual picking for the templates was performed by the EMAN2 E2boxer program. Automated selection was then applied by CryoSPARC.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1e2o

Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 740101

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Atomic model buiding 1

DetailsCOOT was used for structure manipulations, while MOLPROBITY was applied for validation.
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6h05:
Cryo-electron microscopic structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate (2-oxoglutarate) dehydrogenase complex [residues 218-453]

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