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- PDB-9dz8: Catalytic domain of Dihydrolipoamide Succinytransferase -

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Basic information

Entry
Database: PDB / ID: 9dz8
TitleCatalytic domain of Dihydrolipoamide Succinytransferase
ComponentsDihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
KeywordsTRANSFERASE / E2 / 2-oxoglutarate dehydrogenase complex / catalytic domain / tricarboxylic acid cycle
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 BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsCarr, K.D. / Borst, A.J. / Weidle, C.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
B: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
C: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
D: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
E: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
F: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
G: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
H: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
I: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
J: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
K: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
L: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
M: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
N: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
O: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
P: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
Q: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
R: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
S: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
T: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
U: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
V: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
W: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
X: Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex


Theoretical massNumber of molelcules
Total (without water)626,57824
Polymers626,57824
Non-polymers00
Water37,6152088
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex / 2-oxoglutarate dehydrogenase complex component E2


Mass: 26107.420 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A140NDX4, dihydrolipoyllysine-residue succinyltransferase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2088 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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: Dihydrolipoamide Succinyltransferase / Type: COMPLEX
Details: This protein was observed as contaminant in a sample of a two component nanoparticle assembly.
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 1.056 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample was heterogeneous and contamined both DLST and the designed nanoparticle assembly.
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: 295.15 K
Details: Wait time: 7.5 seconds Blot time: 0.5 seconds Blot force: 0 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 5 sec. / Electron dose: 47 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4264
Details: 6211 movies were collected, the best 4264 were used for particle picking and further processing.
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
2SerialEMimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARC4.4.1initial Euler assignment
10cryoSPARC4.4.1final Euler assignment
11cryoSPARC4.4.1classification
12cryoSPARC4.4.13D reconstruction
13ISOLDEmodel refinement
14PHENIXmodel refinement
15Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19033 / Num. of class averages: 76 / Symmetry type: POINT
Atomic model buildingB value: 69.9 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: The 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 ...Details: The 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.
Atomic model buildingDetails: ModelAngelo / Source name: Other / Type: in silico model

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