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- PDB-8orb: 24-meric catalytic domain of dihydrolipoamide acetyltransferase (... -

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Basic information

Entry
Database: PDB / ID: 8orb
Title24-meric catalytic domain of dihydrolipoamide acetyltransferase (E2) of the E. coli pyruvate dehydrogenase complex.
ComponentsDihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complexDihydrolipoyl transacetylase
KeywordsTRANSFERASE / pyruvate dehydrogenase complex / PDHc / E2 / cryo-EM
Function / homology
Function and homology information


pyruvate catabolic process / dihydrolipoyllysine-residue acetyltransferase / dihydrolipoyllysine-residue acetyltransferase activity / acetyl-CoA biosynthetic process from pyruvate / lipoic acid binding / pyruvate dehydrogenase complex / pyruvate metabolic process / acetyltransferase activity / glycolytic process / cytoplasm
Similarity search - Function
Dihydrolipoamide acetyltransferase pyruvate dehydrogenase complex / 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 ...Dihydrolipoamide acetyltransferase pyruvate dehydrogenase complex / 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
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsZdanowicz, R. / Meinhold, S. / Glockshuber, R.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Sci Adv / Year: 2024
Title: Dimerization of a 5-kDa domain defines the architecture of the 5-MDa gammaproteobacterial pyruvate dehydrogenase complex.
Authors: Sarah Meinhold / Rafal Zdanowicz / Christoph Giese / Rudi Glockshuber /
Abstract: The pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). ...The pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E2), we show that two of the three PSBDs in the E2 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E2. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.
History
DepositionApr 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
B: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
C: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
D: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
E: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
F: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
G: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
H: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
I: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
J: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
K: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
L: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
M: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
N: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
O: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
P: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
Q: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
R: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
S: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
T: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
V: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
W: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
X: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
Y: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex


Theoretical massNumber of molelcules
Total (without water)659,78524
Polymers659,78524
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area118930 Å2
ΔGint-913 kcal/mol
Surface area245210 Å2

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Components

#1: Protein ...
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex / Dihydrolipoyl transacetylase / Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex / E2


Mass: 27491.049 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: aceF, b0115, JW0111 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P06959, 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: 24-meric catalytic domain of dihydrolipoamide acetyltransferase (E2) of the E. coli pyruvate dehydrogenase complex
Type: COMPLEX
Details: Wild-type E2 protein was truncated to contain only the catalytic domain
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.66 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
4cryoSPARC3.2CTF correctionPatch CTF Estimation
10cryoSPARC3.2initial Euler assignment
11cryoSPARC3.2final Euler assignment
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 278268 / Symmetry type: POINT

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