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- PDB-3b8k: Structure of the Truncated Human Dihydrolipoyl Acetyltransferase (E2) -

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Basic information

Entry
Database: PDB / ID: 3b8k
TitleStructure of the Truncated Human Dihydrolipoyl Acetyltransferase (E2)
ComponentsDihydrolipoyllysine-residue acetyltransferaseDihydrolipoyl transacetylase
KeywordsTRANSFERASE / central beta-sheet surrounded by five alpha-helices
Function / homology
Function and homology information


dihydrolipoyllysine-residue acetyltransferase / dihydrolipoyllysine-residue acetyltransferase activity / acetyl-CoA biosynthetic process from pyruvate / pyruvate dehydrogenase complex / : / Pyruvate metabolism / Glyoxylate metabolism and glycine degradation / Regulation of pyruvate dehydrogenase (PDH) complex / Signaling by Retinoic Acid / tricarboxylic acid cycle ...dihydrolipoyllysine-residue acetyltransferase / dihydrolipoyllysine-residue acetyltransferase activity / acetyl-CoA biosynthetic process from pyruvate / pyruvate dehydrogenase complex / : / Pyruvate metabolism / Glyoxylate metabolism and glycine degradation / Regulation of pyruvate dehydrogenase (PDH) complex / Signaling by Retinoic Acid / tricarboxylic acid cycle / glucose metabolic process / mitochondrial matrix / intracellular membrane-bounded organelle / mitochondrion / identical protein binding
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 speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.8 Å
AuthorsYu, X. / Hiromasa, Y. / Tsen, H. / Stoops, J.K. / Roche, T.E. / Zhou, Z.H.
CitationJournal: Structure / Year: 2008
Title: Structures of the human pyruvate dehydrogenase complex cores: a highly conserved catalytic center with flexible N-terminal domains.
Authors: Xuekui Yu / Yasuaki Hiromasa / Hua Tsen / James K Stoops / Thomas E Roche / Z Hong Zhou /
Abstract: Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy (cryo- ...Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy (cryo-EM) of the human full-length and truncated E2 (tE2) cores revealed flexible linkers emanating from the edges of trimers of the internal catalytic domains. Using the secondary structure constraints revealed in our 8 A cryo-EM reconstruction and the prokaryotic tE2 atomic structure as a template, we derived a pseudo atomic model of human tE2. The active sites are conserved between prokaryotic tE2 and human tE2. However, marked structural differences are apparent in the hairpin domain and in the N-terminal helix connected to the flexible linker. These permutations away from the catalytic center likely impart structures needed to integrate a second component into the inner core and provide a sturdy base for the linker that holds the pyruvate dehydrogenase for access by the E2-bound regulatory kinase/phosphatase components in humans.
History
DepositionNov 1, 2007Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Assembly

Deposited unit
A: Dihydrolipoyllysine-residue acetyltransferase


Theoretical massNumber of molelcules
Total (without water)25,9381
Polymers25,9381
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Dihydrolipoyllysine-residue acetyltransferase / Dihydrolipoyl transacetylase / E.C.2.3.1.12 / Pyruvate dehydrogenase complex E2 subunit / PDCE2 / E2 / Dihydrolipoamide S- acetyltransferase ...Pyruvate dehydrogenase complex E2 subunit / PDCE2 / E2 / Dihydrolipoamide S- acetyltransferase component of pyruvate dehydrogenase complex / PDC-E2 / 70 kDa mitochondrial autoantigen of primary biliary cirrhosis / PBC / M2 antigen complex 70 kDa subunit


Mass: 25938.164 Da / Num. of mol.: 1 / Fragment: C-TERMINAL CATALYTIC DOMAIN
Source method: isolated from a genetically manipulated source
Details: component of pyruvate dehydrogenase complex, mitochondrial
Source: (gene. exp.) Homo sapiens (human) / Gene: DLAT, DLTA / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P10515, dihydrolipoyllysine-residue acetyltransferase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human tE2 / Type: COMPLEX
Details: Dodecahedron. Human tE2 was prepared from scE2, which contains a PreScission site in the third linker region. Treatment of scE2 with the PreScission protease (Amersham Biosciences) removed ...Details: Dodecahedron. Human tE2 was prepared from scE2, which contains a PreScission site in the third linker region. Treatment of scE2 with the PreScission protease (Amersham Biosciences) removed the N-terminal 319 amino acids. The resulting tE2 was purified by gel filtration with Sephacryl S-300HR. The assembly of the recombinant molecules into fully functional, pentagonal dodecahedral cores was confirmed by analytical ultracentrifugation
Buffer solutionName: PBS / pH: 7.2 / Details: PBS
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: This grid plus sample was kept at -170 deg C during imaging
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Details: flash freezing in liquid ethane

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Electron microscopy imaging

MicroscopyModel: JEOL 2010F / Date: Oct 1, 2003
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 69250 X / Calibrated magnification: 69250 X / Nominal defocus max: 2100 nm / Nominal defocus min: 600 nm / Cs: 1 mm
Specimen holderTemperature: 100 K
Image recordingElectron dose: 12 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Details: 4kx4k

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2IMIRS3D reconstruction
CTF correctionDetails: CTF correction of each image
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: cross-common lines / Resolution: 8.8 Å / Num. of particles: 2432 / Nominal pixel size: 2.17 Å / Actual pixel size: 2.17 Å
Details: Orientation determination and 3D reconstruction were performed using the IMIRS software package on multiprocessor MS Windows XP computer workstations. The orientations were first estimated ...Details: Orientation determination and 3D reconstruction were performed using the IMIRS software package on multiprocessor MS Windows XP computer workstations. The orientations were first estimated from the particle images in the far-from-focus micrographs and refined to about 30- resolution. These orientation parameters were then further refined using the particles in the close-to-focus micrographs.
Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: best fit using the program CHIMERA / Details: REFINEMENT PROTOCOL--rigid body
Atomic model buildingPDB-ID: 1EAA

1eaa


Accession code: 1EAA / Details: Homology model based on PBD ID 1eaa / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms1818 0 0 0 1818

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