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- EMDB-47326: Catalytic domain of Dihydrolipoamide Succinytransferase -

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Basic information

Entry
Database: EMDB / ID: EMD-47326
TitleCatalytic domain of Dihydrolipoamide Succinytransferase
Map dataSharpened map using DeepEMhancer
Sample
  • Complex: Dihydrolipoamide Succinyltransferase
    • Protein or peptide: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
  • Ligand: water
KeywordsE2 / 2-oxoglutarate dehydrogenase complex / catalytic domain / tricarboxylic acid cycle / TRANSFERASE
Function / homology
Function and homology information


L-lysine catabolic process to acetyl-CoA via saccharopine / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / oxoglutarate dehydrogenase complex / tricarboxylic acid cycle / cytosol
Similarity search - Function
: / Dihydrolipoamide succinyltransferase / 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) ...: / Dihydrolipoamide succinyltransferase / 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 succinyltransferase component of 2-oxoglutarate dehydrogenase complex
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsCarr KD / Borst AJ / Weidle C
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation United States
CitationJournal: J Struct Biol X / Year: 2025
Title: Protein identification using Cryo-EM and artificial intelligence guides improved sample purification.
Authors: Kenneth D Carr / Dane Evan D Zambrano / Connor Weidle / Alex Goodson / Helen E Eisenach / Harley Pyles / Alexis Courbet / Neil P King / Andrew J Borst /
Abstract: Protein purification is essential in protein biochemistry, structural biology, and protein design, enabling the determination of protein structures, the study of biological mechanisms, and the ...Protein purification is essential in protein biochemistry, structural biology, and protein design, enabling the determination of protein structures, the study of biological mechanisms, and the characterization of both natural and de novo designed proteins. However, standard purification strategies often encounter challenges, such as unintended co-purification of contaminants alongside the target protein. This issue is particularly problematic for self-assembling protein nanomaterials, where unexpected geometries may reflect novel assembly states, cross-contamination, or native proteins originating from the expression host. Here, we used an automated structure-to-sequence pipeline to first identify an unknown co-purifying protein found in several purified designed protein samples. By integrating cryo-electron microscopy (Cryo-EM), ModelAngelo's sequence-agnostic model-building, and Protein BLAST, we identified the contaminant as dihydrolipoamide succinyltransferase (DLST). This identification was validated through comparisons with DLST structures in the Protein Data Bank, AlphaFold 3 predictions based on the DLST sequence from our E. coli expression vector, and traditional biochemical methods. The identification informed subsequent modifications to our purification protocol, which successfully excluded DLST from future preparations. To explore the potential broader utility of this approach, we benchmarked four computational methods for DLST identification across varying resolution ranges. This study demonstrates the successful application of a structure-to-sequence protein identification workflow, integrating Cryo-EM, ModelAngelo, Protein BLAST, and AlphaFold 3 predictions, to identify and ultimately help guide the removal of DLST from sample purification efforts. It highlights the potential of combining Cryo-EM with AI-driven tools for accurate protein identification and addressing purification challenges across diverse contexts in protein science.
History
DepositionOct 15, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_47326.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map using DeepEMhancer
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.028140226 - 1.6010301
Average (Standard dev.)0.00071355194 (±0.018694753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 503.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dihydrolipoamide Succinyltransferase

EntireName: Dihydrolipoamide Succinyltransferase
Components
  • Complex: Dihydrolipoamide Succinyltransferase
    • Protein or peptide: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
  • Ligand: water

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Supramolecule #1: Dihydrolipoamide Succinyltransferase

SupramoleculeName: Dihydrolipoamide Succinyltransferase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: This protein was observed as contaminant in a sample of a two component nanoparticle assembly.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Molecular weightTheoretical: 1.056 MDa

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

MacromoleculeName: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: dihydrolipoyllysine-residue succinyltransferase
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 26.10742 KDa
SequenceString: ARSEKRVPMT RLRKRVAERL LEAKNSTAML TTFNEVNMKP IMDLRKQYGE AFEKRHGIRL GFMSFYVKAV VEALKRYPEV NASIDGDDV VYHNYFDVSM AVSTPRGLVT PVLRDVDTLG MADIEKKIKE LAVKGRDGKL TVEDLTGGNF TITNGGVFGS L MSTPIINP ...String:
ARSEKRVPMT RLRKRVAERL LEAKNSTAML TTFNEVNMKP IMDLRKQYGE AFEKRHGIRL GFMSFYVKAV VEALKRYPEV NASIDGDDV VYHNYFDVSM AVSTPRGLVT PVLRDVDTLG MADIEKKIKE LAVKGRDGKL TVEDLTGGNF TITNGGVFGS L MSTPIINP PQSAILGMHA IKDRPMAVNG QVEILPMMYL ALSYDHRLID GRESVGFLVT IKELLEDPTR LLLDV

UniProtKB: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 2088 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.3 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 40 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
Details: Wait time: 7.5 seconds Blot time: 0.5 seconds Blot force: 0 seconds.
DetailsThis sample was heterogeneous and contamined both DLST and the designed nanoparticle assembly.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 4264 / Average exposure time: 5.0 sec. / Average electron dose: 47.0 e/Å2
Details: 6211 movies were collected, the best 4264 were used for particle picking and further processing.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: ab-initio
Final reconstructionNumber classes used: 76 / Applied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 19033
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final 3D classificationNumber classes: 76 / Avg.num./class: 250 / Software - Name: cryoSPARC (ver. 4.4.1)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: ModelAngelo
DetailsThe final model was built to density using the UniProt sequence in ModelAngelo. Further refinement of the model to the density was performed using ISOLDE in ChimeraX, Coot, Phenix. Waters were built to one chain and then that chain and water network were rebuilt in ChimeraX using symmetry.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 69.9 / Target criteria: Cross-correlation coefficient
Output model

PDB-9dz8:
Catalytic domain of Dihydrolipoamide Succinytransferase

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