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- EMDB-12104: Cryo-EM structure of the dihydrolipoyl transacetylase cubic core ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12104
TitleCryo-EM structure of the dihydrolipoyl transacetylase cubic core of the E. coli pyruvate dehydrogenase complex including lipoyl domains
Map data
Sample
  • Complex: dihydrolipoyl transacetylase cubic core of the pyruvate dehydrogenase complex
    • Protein or peptide: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complexDihydrolipoyl transacetylase
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) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsSkerlova J / Stenmark P
Funding support Sweden, European Union, 2 items
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, 2020-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.3
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  • Surface view with fitted model
  • Atomic models: PDB-7b9k
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12104.png

Downloads & links

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Map

FileDownload / File: emd_12104.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-1.2099564 - 2.6830359
Average (Standard dev.)0.007123699 (±0.10626714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z312.000312.000312.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-1.2102.6830.007

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Supplemental data

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Mask #1

Fileemd_12104_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12104_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12104_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dihydrolipoyl transacetylase cubic core of the pyruvate dehydroge...

EntireName: dihydrolipoyl transacetylase cubic core of the pyruvate dehydrogenase complex
Components
  • Complex: dihydrolipoyl transacetylase cubic core of the pyruvate dehydrogenase complex
    • Protein or peptide: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complexDihydrolipoyl transacetylase

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Supramolecule #1: dihydrolipoyl transacetylase cubic core of the pyruvate dehydroge...

SupramoleculeName: dihydrolipoyl transacetylase cubic core of the pyruvate dehydrogenase complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12

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Macromolecule #1: Dihydrolipoyllysine-residue acetyltransferase component of pyruva...

MacromoleculeName: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
type: protein_or_peptide / ID: 1 / Details: Lysine 248 covalently modified by dihydrolipoate / Number of copies: 24 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue acetyltransferase
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 66.363203 KDa
SequenceString: MAIEIKVPDI GADEVEITEI LVKVGDKVEA EQSLITVEGD KASMEVPSPQ AGIVKEIKVS VGDKTQTGAL IMIFDSADGA ADAAPAQAE EKKEAAPAAA PAAAAAKDVN VPDIGSDEVE VTEILVKVGD KVEAEQSLIT VEGDKASMEV PAPFAGTVKE I KVNVGDKV ...String:
MAIEIKVPDI GADEVEITEI LVKVGDKVEA EQSLITVEGD KASMEVPSPQ AGIVKEIKVS VGDKTQTGAL IMIFDSADGA ADAAPAQAE EKKEAAPAAA PAAAAAKDVN VPDIGSDEVE VTEILVKVGD KVEAEQSLIT VEGDKASMEV PAPFAGTVKE I KVNVGDKV STGSLIMVFE VAGEAGAAAP AAKQEAAPAA APAPAAGVKE VNVPDIGGDE VEVTEVMVKV GDKVAAEQSL IT VEGD(LA2)AS MEVPAPFAGV VKELKVNVGD KVKTGSLIMI FEVEGAAPAA APAKQEAAAP APAAKAEAPA AAPAAKAEG KSEFAENDAY VHATPLIRRL AREFGVNLAK VKGTGRKGRI LREDVQAYVK EAIKRAEAAP AATGGGIPGM LPWPKVDFSK FGEIEEVEL GRIQKISGAN LSRNWVMIPH VTHFDKTDIT ELEAFRKQQN EEAAKRKLDV KITPVVFIMK AVAAALEQMP R FNSSLSED GQRLTLKKYI NIGVAVDTPN GLVVPVFKDV NKKGIIELSR ELMTISKKAR DGKLTAGEMQ GGCFTISSIG GL GTTHFAP IVNAPEVAIL GVSKSAMEPV WNGKEFVPRL MLPISLSFDH RVIDGADGAR FITIINNTLS DIRRLVM

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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 #1

Preparation ID1
Concentration0.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHepes
150.0 mMsodium chlorideNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Film type ID: 1 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliters of sample, blot force 0, 15 second wait time, 3 second blot time.

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Sample preparation #2

Preparation ID2
Concentration1.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHepes
150.0 mMsodium chlorideNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Film type ID: 1 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliters of sample, blot force 0, 15 second wait time, 3 second blot time.

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Sample preparation #3

Preparation ID3
Concentration0.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHepes
150.0 mMsodium chlorideNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Film type ID: 1 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliters of sample, blot force 0, 15 second wait time, 3 second blot time.

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Electron microscopy #1

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Microscopy ID1
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3708 / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Microscopy ID1
Image recordingImage recording ID: 2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3558 / Average electron dose: 28.11 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~~

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Microscopy ID1
Image recordingImage recording ID: 3 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 12867 / Average electron dose: 28.11 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 799790 / Details: automated template picking in CryoSPARC
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final 3D classificationNumber classes: 2 / Avg.num./class: 21133 / Software - Name: cryoSPARC (ver. 2.15)
Details: 12832 particles (class 1) discarded and 29434 particles (class 2) used for the final reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 29434
Image recording ID1
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

4n72

,

1qjo

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7b9k:
Cryo-EM structure of the dihydrolipoyl transacetylase cubic core of the E. coli pyruvate dehydrogenase complex including lipoyl domains

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