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- PDB-9mu6: Structure of native Drosophila melanogaster DLST -

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Basic information

Entry
Database: PDB / ID: 9mu6
TitleStructure of native Drosophila melanogaster DLST
ComponentsDihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial
KeywordsGENE REGULATION / chromatin / succinyltransferase / DLST
Function / homology
Function and homology information


Glycine degradation / OGDH complex synthesizes succinyl-CoA from 2-OG / OADH complex synthesizes glutaryl-CoA from 2-OA / Protein lipoylation / oxoadipate dehydrogenase complex / L-lysine catabolic process to acetyl-CoA via saccharopine / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / oxoglutarate dehydrogenase complex / cellular respiration ...Glycine degradation / OGDH complex synthesizes succinyl-CoA from 2-OG / OADH complex synthesizes glutaryl-CoA from 2-OA / Protein lipoylation / oxoadipate dehydrogenase complex / L-lysine catabolic process to acetyl-CoA via saccharopine / dihydrolipoyllysine-residue succinyltransferase / dihydrolipoyllysine-residue succinyltransferase activity / oxoglutarate dehydrogenase complex / cellular respiration / tricarboxylic acid cycle / Z disc / mitochondrion
Similarity search - Function
: / Dihydrolipoamide succinyltransferase / 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, mitochondrial
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsVenette-Smith, N.L. / Vishwakarma, R.K. / Dollinger, R. / Schultz, J. / Venkatakrishnan, V. / Babitzke, P. / Anand, G. / Gilmour, D.S. / Armache, J.-P. / Murakami, K.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM047477 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098399 United States
CitationJournal: To Be Published
Title: Structural Characterization of Native RNA Polymerase II Transcription Complexes in Drosophila melanogaster
Authors: Venette-Smith, N.L. / Vishwakarma, R.K. / Dollinger, R. / Schultz, J. / Venkatakrishnan, V. / Babitzke, P. / Anand, G. / Gilmour, D.S. / Armache, J.-P. / Murakami, K.
History
DepositionJan 13, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)618,14524
Polymers618,14524
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial / E2K


Mass: 25756.025 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly)
References: UniProt: Q9VGQ1, dihydrolipoyllysine-residue succinyltransferase
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: Natively purified DLST from Drosophila melanogaster / Type: COMPLEX
Details: This entry represents a native dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial, purified from Drosophila melanogaster embryos
Entity ID: all / Source: NATURAL
Molecular weightValue: 650 kDa/nm / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Buffer solutionpH: 7.5
Details: 10 mM HEPES-HCl (pH = 7.5), 150 mM NaCl, 5% glycerol, 1 mM EDTA, 350 ug/mL 3x FLAG peptide, 1/1000th protease inhibitor
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
210 mMHEPESHEPES-HCl1
35 percentGlycerolGlycerol1
41 mMEDTAEDTA1
5350 ug/mLFLAG peptideFLAG1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: double FLAG-tagged Pol II subunit Rpb1 was used for purification of Pol II complexes. We aimed to purify Pol II in tandem with nucleosomes. DLST was co-purified with this sample
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.65 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 31103
Details: Although the data was collected, motion-corrected and CTF estimated using the pixel size of 0.944, all the subsequent data processing was performed using pixel size of 1.1538. This was ...Details: Although the data was collected, motion-corrected and CTF estimated using the pixel size of 0.944, all the subsequent data processing was performed using pixel size of 1.1538. This was achieved by extracting particles in box size 440x440 and Fourier-cropping it to box size 360x360
Image scansSampling size: 0.944 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.1particle selection
2EPU3.4image acquisition
4cryoSPARC4.3.1CTF correction
7UCSF ChimeraX1.8model fitting
9cryoSPARC4.3.1initial Euler assignment
10cryoSPARC4.3.1final Euler assignment
12cryoSPARC4.3.13D reconstruction
13Coot0.9.8.7model refinement
14PHENIX1.21.5207model refinement
CTF correctionDetails: 'Patch CTF Estimation' in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 70106
Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (70,106) is the starting number of particles after initial cleanup and 3D ...Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (70,106) is the starting number of particles after initial cleanup and 3D classification of the data.
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23543 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 50 / Protocol: AB INITIO MODEL
Details: The model was built without a template. AlphaFold2 models for individual subunits were inserted into the density, and further optimized in the density using rigid-body fit. Then, in Coot, ...Details: The model was built without a template. AlphaFold2 models for individual subunits were inserted into the density, and further optimized in the density using rigid-body fit. Then, in Coot, the models were Real-space refined. For the final model optimization, phenix.real_space_refine was used
Atomic model buildingAccession code: Q9VGQ1 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00543944
ELECTRON MICROSCOPYf_angle_d0.96859328
ELECTRON MICROSCOPYf_dihedral_angle_d5.2556192
ELECTRON MICROSCOPYf_chiral_restr0.0626840
ELECTRON MICROSCOPYf_plane_restr0.0087680

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