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- PDB-7b9k: Cryo-EM structure of the dihydrolipoyl transacetylase cubic core ... -

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Basic information

Entry
Database: PDB / ID: 7b9k
TitleCryo-EM structure of the dihydrolipoyl transacetylase cubic core of the E. coli pyruvate dehydrogenase complex including lipoyl domains
ComponentsDihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complexDihydrolipoyl transacetylase
KeywordsTRANSFERASE / Multienzyme complexes / oxoacid dehydrogenase complexes / lipoic acid / dihydrolipoyllysine
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: 3.16 Å
AuthorsSkerlova, J. / Stenmark, P.
Funding support Sweden, European Union, 2items
OrganizationGrant numberCountry
Swedish Research Council2018-03406 Sweden
European Regional Development Fund"IOCB Mobility" CZ.02.2.69/0.0/0.0/16_027/0008477European Union
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion.
Authors: Jana Škerlová / Jens Berndtsson / Hendrik Nolte / Martin Ott / Pål Stenmark /
Abstract: The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely ...The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.
History
DepositionDec 14, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code

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Structure visualization

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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
U: 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


Theoretical massNumber of molelcules
Total (without water)1,592,71724
Polymers1,592,71724
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 66363.203 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Details: Lysine 248 covalently modified by dihydrolipoate / Source: (natural) Escherichia coli (strain K12) (bacteria) / Strain: K12
References: UniProt: P06959, dihydrolipoyllysine-residue acetyltransferase
Has ligand of interestY

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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: dihydrolipoyl transacetylase cubic core of the pyruvate dehydrogenase complex
Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Buffer solution
IDSpecimen-IDpH
117.5
227.5
337.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2150 mMsodium chlorideNaClSodium chloride1
320 mMHepes2
4150 mMsodium chlorideNaClSodium chloride2
520 mMHepes3
6150 mMsodium chlorideNaClSodium chloride3
Specimen
IDConc. (mg/ml)Experiment-IDEmbedding appliedShadowing appliedStaining appliedVitrification applied
10.61NONONOYES
21.41NONONOYES
30.71NONONOYES
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid typeDetails
11COPPER300Quantifoil R2/2
22COPPER300Quantifoil R1.2/1.3
33COPPER300Quantifoil R2/2grid coated with a 0.2 mg/ml suspension of graphene oxide after glow discharge
Vitrification

Chamber temperature: 277 K / Cryogen name: ETHANE / Entry-ID: 7B9K / Humidity: 100 % / Instrument: FEI VITROBOT MARK IV

IDSpecimen-IDDetails
113 microliters of sample, blot force 0, 15 second wait time, 3 second blot time
223 microliters of sample, blot force 0, 15 second wait time, 3 second blot time
332.5 microliters of sample, blot force 0, 15 second wait time, 3 second blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm

IDSpecimen-ID
11
22
33
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector modelNum. of grids imagedNum. of real images
1130COUNTINGGATAN K2 QUANTUM (4k x 4k)13708
2228.11COUNTINGGATAN K2 QUANTUM (4k x 4k)13558
3328.11COUNTINGGATAN K2 QUANTUM (4k x 4k)112867

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2.15particle selection
2EPUimage acquisition
4cryoSPARC2.15CTF correction
7PHENIX1.16model fittingdock_in_map
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
11cryoSPARC2.15classification
12cryoSPARC2.153D reconstruction
13PHENIX1.16model refinement
14REFMAC5.8.0232model refinementfrom CCP-EM 1.4.1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 799790 / Details: automated template picking in CryoSPARC
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29434 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
14N72

4n72

1
21QJO

1qjo

1

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