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- EMDB-26981: Cryo-EM structure of Mtb Lpd bound to inhibitor complex with 2-((... -

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Basic information

Entry
Database: EMDB / ID: EMD-26981
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
Map dataDensity-modified map generated using phenix.cryoem.resolve
Sample
  • Complex: 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
    • Protein or peptide: Dihydrolipoyl dehydrogenase
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: 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
  • Ligand: water
Keywordsflavoprotein / glycolysis / redox-active center / OXIDOREDUCTASE-INHIBITOR complex
Function / homology
Function and homology information


Cell redox homeostasis / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase activity / NADH binding / pyruvate dehydrogenase complex / disulfide oxidoreductase activity / 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 / dihydrolipoyl dehydrogenase / dihydrolipoyl dehydrogenase activity / NADH binding / pyruvate dehydrogenase complex / disulfide oxidoreductase activity / 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
Dihydrolipoyl dehydrogenase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.17 Å
AuthorsKochanczyk T / Arango N / Lima CD
Funding support United States, 3 items
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 CD
History
DepositionMay 13, 2022-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26981.png

Downloads & links

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Map

FileDownload / File: emd_26981.map.gz / Format: CCP4 / Size: 4.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity-modified map generated using phenix.cryoem.resolve
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 1.5
Minimum - Maximum-8.266245 - 20.0959
Average (Standard dev.)-0.000000000018538 (±0.87924695)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin131145142
Dimensions12389101
Spacing10112389
CellA: 107.464005 Å / B: 130.872 Å / C: 94.696 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Raw map

Fileemd_26981_additional_1.map
AnnotationRaw map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1

Fileemd_26981_half_map_1.map
Annotationhalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 2

Fileemd_26981_half_map_2.map
Annotationhalf-map 2
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 dehydrogenase in complex with 2-((2-cyano-N,5-dimet...

EntireName: 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
Components
  • Complex: 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
    • Protein or peptide: Dihydrolipoyl dehydrogenase
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: 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
  • Ligand: water

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Supramolecule #1: Dihydrolipoyl dehydrogenase in complex with 2-((2-cyano-N,5-dimet...

SupramoleculeName: 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 / ID: 1 / Parent: 0 / Macromolecule list: #1
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)
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 99 KDa

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Macromolecule #1: Dihydrolipoyl dehydrogenase

MacromoleculeName: Dihydrolipoyl dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: dihydrolipoyl dehydrogenase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 49.437035 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSMTHYDVVV LGAGPGGYVA AIRAAQLGLS TAIVEPKYWG GVCLNVGCIP SKALLRNAEL VHIFTKDAKA FGISGEVTFD YGIAYDRSR KVAEGRVAGV HFLMKKNKIT EIHGYGTFAD ANTLLVDLND GGTESVTFDN AIIATGSSTR LVPGTSLSAN V VTYEEQIL ...String:
GSMTHYDVVV LGAGPGGYVA AIRAAQLGLS TAIVEPKYWG GVCLNVGCIP SKALLRNAEL VHIFTKDAKA FGISGEVTFD YGIAYDRSR KVAEGRVAGV HFLMKKNKIT EIHGYGTFAD ANTLLVDLND GGTESVTFDN AIIATGSSTR LVPGTSLSAN V VTYEEQIL SRELPKSIII AGAGAIGMEF GYVLKNYGVD VTIVEFLPRA LPNEDADVSK EIEKQFKKLG VTILTATKVE SI ADGGSQV TVTVTKDGVA QELKAEKVLQ AIGFAPNVEG YGLDKAGVAL TDRKAIGVDD YMRTNVGHIY AIGDVNGLLQ LAH VAEAQG VVAAETIAGA ETLTLGDHRM LPRATFCQPN VASFGLTEQQ ARNEGYDVVV AKFPFTANAK AHGVGDPSGF VKLV ADAKH GELLGGHLVG HDVAELLPEL TLAQRWDLTA SELARNVHTH PTMSEALQEC FHGLVGHMIN F

UniProtKB: Dihydrolipoyl dehydrogenase

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Macromolecule #2: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 2 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #3: N~2~-(2-cyano-5-methyl-1H-indole-7-sulfonyl)-N~2~-methyl-N-[4-(ox...

MacromoleculeName: 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
type: ligand / ID: 3 / Number of copies: 2 / Formula: OU6
Molecular weightTheoretical: 495.551 Da
Chemical component information

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

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 665 / 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

Concentration3 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCl, pH 8.0, 87.5 mM NaCl, 0.05% IGEPAL
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: Temperature 22 C, humidity 100%, Wait time 8s, Blot time: 3.5s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 10748 / Average exposure time: 4.0 sec. / Average electron dose: 70.66 e/Å2
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: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1177958
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0) / Details: Non-uniform refinement in cryoSPARC was used.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0) / Details: Non-uniform refinement in cryoSPARC was used.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7kmy


Chain - Chain ID: AB / Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: PHENIX (ver. 1.20.1-44487)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-8ct4:
Cryo-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

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