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- PDB-8ct4: Cryo-EM structure of Mtb Lpd bound to inhibitor complex with 2-((... -

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Basic information

Entry
Database: PDB / ID: 8ct4
TitleCryo-EM structure of Mtb Lpd bound to inhibitor complex with 2-((2-cyano-N,5-dimethyl-1H-indole)-7-sulfonamido)-N-(4-(oxetan-3-yl)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-7-yl)acetamide
ComponentsDihydrolipoyl dehydrogenase
KeywordsOXIDOREDUCTASE/INHIBITOR / flavoprotein / glycolysis / redox-active center / OXIDOREDUCTASE-INHIBITOR complex
Function / homology
Function and homology information


Cell redox homeostasis / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase (NADH) activity / NADH binding / disulfide oxidoreductase activity / pyruvate dehydrogenase complex / 2-oxoglutarate metabolic process / pyruvate metabolic process / zymogen binding / oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor ...Cell redox homeostasis / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase (NADH) activity / NADH binding / disulfide oxidoreductase activity / pyruvate dehydrogenase complex / 2-oxoglutarate metabolic process / pyruvate metabolic process / zymogen binding / oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor / antioxidant activity / Prevention of phagosomal-lysosomal fusion / cell redox homeostasis / flavin adenine dinucleotide binding / extracellular region / plasma membrane / cytosol
Similarity search - Function
Dihydrolipoamide dehydrogenase / : / Pyridine nucleotide-disulphide oxidoreductase, class I / Pyridine nucleotide-disulphide oxidoreductase, class I, active site / Pyridine nucleotide-disulphide oxidoreductases class-I active site. / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain / FAD/NAD-linked reductase, dimerisation domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / Chem-OU6 / Dihydrolipoyl dehydrogenase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.17 Å
AuthorsKochanczyk, T. / Arango, N. / Lima, C.D.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118080 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748 United States
CitationJournal: Not published
Title: Cryo-EM structure of Mtb Lpd bound to the inhibitor 2-((2-cyano-N,5-dimethyl-1H-indole)-7-sulfonamido)-N-(4-(oxetan-3-yl)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-7-yl)acetamide at 2.17 Angstrom resolution
Authors: Kochanczyk, T. / Arango, N. / Michino, M. / Sun, S. / Ginn, J. / Bryk, R. / Nathan, C. / Lima, C.D.
History
DepositionMay 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 7, 2023Group: Database references / Refinement description / Category: citation / pdbx_initial_refinement_model / Item: _citation.journal_abbrev / _citation.unpublished_flag
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydrolipoyl dehydrogenase
B: Dihydrolipoyl dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,4366
Polymers98,8742
Non-polymers2,5624
Water11,980665
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Dimer also by gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Dihydrolipoyl dehydrogenase / LPD / Component of peroxynitrite reductase/peroxidase complex / Component of PNR/P / ...LPD / Component of peroxynitrite reductase/peroxidase complex / Component of PNR/P / Dihydrolipoamide dehydrogenase / E3 component of alpha-ketoacid dehydrogenase complexes


Mass: 49437.035 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Strain: ATCC 25618 / H37Rv / Gene: lpdC, lpd, Rv0462, MTV038.06 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WHH9, dihydrolipoyl dehydrogenase
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Chemical ChemComp-OU6 / N~2~-(2-cyano-5-methyl-1H-indole-7-sulfonyl)-N~2~-methyl-N-[4-(oxetan-3-yl)-3,4-dihydro-2H-1,4-benzoxazin-7-yl]glycinamide


Mass: 495.551 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H25N5O5S / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 665 / Source method: isolated from a natural source / Formula: H2O
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 dehydrogenase in complex with 2-((2-cyano-N,5-dimethyl-1H-indole)-7-sulfonamido)-N-(4-(oxetan-3-yl)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-7-yl)acetamide
Type: COMPLEX
Details: 2-((2-cyano-N,5-dimethyl-1H-indole)-7-sulfonamido)-N-(4-(oxetan-3-yl)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-7-yl)acetamide provided by the Tri-Institutional Therapeutics Discovery Institute ...Details: 2-((2-cyano-N,5-dimethyl-1H-indole)-7-sulfonamido)-N-(4-(oxetan-3-yl)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-7-yl)acetamide provided by the Tri-Institutional Therapeutics Discovery Institute (tritdi.org) contact: John Ginn (jginn@tritdi.org)
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.099 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: ATCC 25618 / H37Rv
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl, pH 8.0, 87.5 mM NaCl, 0.05% IGEPAL
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: Temperature 22 C, humidity 100%, Wait time 8s, Blot time: 3.5s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 70.66 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10748

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Processing

EM software
IDNameVersionCategory
4cryoSPARC3.1.0CTF correction
5RELION3.1.2CTF correction
8PHENIX1.20.1-44487model fitting
10Coot0.9model refinement
11PHENIX1.20.1-44487model refinement
12cryoSPARC3.1.0initial Euler assignment
13cryoSPARC3.1.0final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1177958 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 7KMY

7kmy


Pdb chain-ID: AB / Accession code: 7KMY / Source name: PDB / Type: experimental model

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