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- PDB-8y6p: Structure of the auto-inhibited Dark monomer -

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Basic information

Entry
Database: PDB / ID: 8y6p
TitleStructure of the auto-inhibited Dark monomer
ComponentsApaf-1 related killer DARK
KeywordsAPOPTOSIS / Dark / cryo-EM
Function / homology
Function and homology information


negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / salivary gland histolysis / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / compound eye retinal cell programmed cell death / central nervous system formation ...negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / salivary gland histolysis / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / compound eye retinal cell programmed cell death / central nervous system formation / positive regulation of apoptotic process involved in development / chaeta development / sperm individualization / apoptosome / autophagic cell death / Neutrophil degranulation / S-adenosylmethionine cycle / CARD domain binding / programmed cell death / triglyceride homeostasis / response to starvation / dendrite morphogenesis / response to gamma radiation / neuron cellular homeostasis / ADP binding / apoptotic process / structural molecule activity / ATP binding / identical protein binding
Similarity search - Function
: / : / Dark, CARD domain / Dark, winged-helix domain / APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / WD40 repeats / WD40 repeat ...: / : / Dark, CARD domain / Dark, winged-helix domain / APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / WD40 repeats / WD40 repeat / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Apoptosome protein Dark
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsTian, L. / Li, Y. / Shi, Y.
Funding support1items
OrganizationGrant numberCountry
Other private
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Dark and Dronc activation in .
Authors: Lu Tian / Yini Li / Yigong Shi /
Abstract: The onset of apoptosis is characterized by a cascade of caspase activation, where initiator caspases are activated by a multimeric adaptor complex known as the apoptosome. In , the initiator caspase ...The onset of apoptosis is characterized by a cascade of caspase activation, where initiator caspases are activated by a multimeric adaptor complex known as the apoptosome. In , the initiator caspase Dronc undergoes autocatalytic activation in the presence of the Dark apoptosome. Despite rigorous investigations, the activation mechanism for Dronc remains elusive. Here, we report the cryo-EM structures of an auto-inhibited Dark monomer and a single-layered, multimeric Dark/Dronc complex. Our biochemical analysis suggests that the auto-inhibited Dark oligomerizes upon binding to Dronc, which is sufficient for the activation of both Dark and Dronc. In contrast, the previously observed double-ring Dark apoptosome may represent a non-functional or "off-pathway" conformation. These findings expand our understanding on the molecular mechanism of apoptosis in .
History
DepositionFeb 3, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
Q: Apaf-1 related killer DARK


Theoretical massNumber of molelcules
Total (without water)166,6041
Polymers166,6041
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 Apaf-1 related killer DARK / Apaf-1/CED-4-related caspase activator Dapaf-1L / Cell death protein HAC-1 / Death-associated APAF1- ...Apaf-1/CED-4-related caspase activator Dapaf-1L / Cell death protein HAC-1 / Death-associated APAF1-related killer / isoform B / FI21208p1


Mass: 166603.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Dark, anon-53Fa, Apaf, APAF-1, Apaf-1, apaf-1, APAF1, Apaf1, apaf1, arc, ARK, Ark, ark, Ark-RB, D-Apaf-1, dapaf, Dapaf-1, dAPAF-1, dApaf-1, dapaf-1, dApaf-1/DARK/HAC-1, Dapaf-1/HAC-1, dapaf-1L, ...Gene: Dark, anon-53Fa, Apaf, APAF-1, Apaf-1, apaf-1, APAF1, Apaf1, apaf1, arc, ARK, Ark, ark, Ark-RB, D-Apaf-1, dapaf, Dapaf-1, dAPAF-1, dApaf-1, dapaf-1, dApaf-1/DARK/HAC-1, Dapaf-1/HAC-1, dapaf-1L, dapaf-1S, dApaf1, DARK, dArk, dark, Dark/Apaf-I, dark/dapaf-1/hac-1, Dark/Dapaf-1/HAC1, Dark/Hac-1/dApaf-1, dark/hac-1/dapaf-1, Dark/Hac-1/dApaf1, Dmel\CG6829, Hac-1, hac-1, Hac-1/Dark, Hac1, hac1, l(2)SH0173, T1, CG6829, Dmel_CG6829
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7KLI1

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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: Structure of the auto-inhibited Dark monomer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105018 / Symmetry type: POINT

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