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- PDB-3j9k: Structure of Dark apoptosome in complex with Dronc CARD domain -

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Basic information

Entry
Database: PDB / ID: 3j9k
TitleStructure of Dark apoptosome in complex with Dronc CARD domain
Components
  • Apaf-1 related killer DARK
  • Caspase Nc
KeywordsAPOPTOSIS / programmed cell death / caspase activation
Function / homology
Function and homology information


hemocyte development / nurse cell apoptotic process / TP53 Regulates Transcription of Caspase Activators and Caspases / negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / head involution / Formation of apoptosome / embryonic development via the syncytial blastoderm / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death ...hemocyte development / nurse cell apoptotic process / TP53 Regulates Transcription of Caspase Activators and Caspases / negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / head involution / Formation of apoptosome / embryonic development via the syncytial blastoderm / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / central nervous system formation / metamorphosis / compound eye development / chaeta development / sperm individualization / apoptosome / autophagic cell death / programmed cell death involved in cell development / Neutrophil degranulation / CARD domain binding / programmed necrotic cell death / S-adenosylmethionine cycle / : / programmed cell death / triglyceride homeostasis / zymogen activation / dendrite morphogenesis / neuron remodeling / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / protein autoprocessing / ectopic germ cell programmed cell death / determination of adult lifespan / response to gamma radiation / central nervous system development / ADP binding / neuron cellular homeostasis / endopeptidase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / positive regulation of apoptotic process / negative regulation of cell population proliferation / cysteine-type endopeptidase activity / apoptotic process / protein homodimerization activity / ATP binding / identical protein binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
: / : / Dark, CARD domain / Dark, winged-helix domain / APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site ...: / : / Dark, CARD domain / Dark, winged-helix domain / APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. / Caspase family p10 domain profile. / Peptidase C14A, caspase catalytic domain / Caspase, interleukin-1 beta converting enzyme (ICE) homologues / Peptidase C14, p20 domain / Caspase family p20 domain profile. / : / Caspase domain / Caspase-like domain superfamily / Death-like domain superfamily / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Apaf-1 related killer DARK / Caspase Dronc
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsPang, Y. / Bai, X. / Yan, C. / Hao, Q. / Chen, Z. / Wang, J. / Scheres, S.H.W. / Shi, Y.
CitationJournal: Genes Dev / Year: 2015
Title: Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila.
Authors: Yuxuan Pang / Xiao-chen Bai / Chuangye Yan / Qi Hao / Zheqin Chen / Jia-Wei Wang / Sjors H W Scheres / Yigong Shi /
Abstract: Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is ...Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome.
History
DepositionFeb 4, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 25, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.2Feb 21, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Apaf-1 related killer DARK
B: Caspase Nc
C: Apaf-1 related killer DARK
D: Caspase Nc
E: Apaf-1 related killer DARK
F: Caspase Nc
G: Apaf-1 related killer DARK
H: Caspase Nc
I: Apaf-1 related killer DARK
J: Caspase Nc
K: Apaf-1 related killer DARK
L: Caspase Nc
M: Apaf-1 related killer DARK
N: Caspase Nc
O: Apaf-1 related killer DARK
P: Caspase Nc
Q: Apaf-1 related killer DARK
R: Caspase Nc
S: Apaf-1 related killer DARK
T: Caspase Nc
U: Apaf-1 related killer DARK
V: Caspase Nc
W: Apaf-1 related killer DARK
X: Caspase Nc
Y: Apaf-1 related killer DARK
Z: Caspase Nc
a: Apaf-1 related killer DARK
b: Caspase Nc
c: Apaf-1 related killer DARK
d: Caspase Nc
e: Apaf-1 related killer DARK
f: Caspase Nc
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,630,13648
Polymers2,623,30132
Non-polymers6,83516
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Apaf-1 related killer DARK / Apaf-1-related-killer / isoform B / Apaf-1/CED-4-related caspase activator Dapaf-1L / Cell death ...Apaf-1-related-killer / isoform B / Apaf-1/CED-4-related caspase activator Dapaf-1L / Cell death protein HAC-1 / FI21208p1


Mass: 112749.156 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: Q7KLI1
#2: Protein
Caspase Nc / NEDD2-like caspase DRONC / Caspase Nc subunit 1 / Caspase Nc subunit 2 / Dronc-CARD


Mass: 51207.145 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly)
References: UniProt: Q9XYF4, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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

Component
IDNameTypeParent-ID
1Dark apoptosome in complex with Dronc-CARDCOMPLEX0
2Dark apoptosome1
3Dronc-CARD1
Molecular weightValue: 3 MDa / Experimental value: NO
Buffer solutionName: 25 mM Tris, pH 8.0, 150 mM NaCl, 5 mM DTT / pH: 8 / Details: 25 mM Tris, pH 8.0, 150 mM NaCl, 5 mM DTT
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: glow-discharged holey carbon grids (Quantifoil CuR2/2) with home-made continuous carbon film
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temp: 85 K / Humidity: 100 %
Details: Blot for 2 seconds before plunging into liquid ethane (FEI VITROBOT MARK II).
Method: Blot for 2 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jan 8, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 78000 X / Calibrated magnification: 104748 X / Nominal defocus max: 6400 nm / Nominal defocus min: 1400 nm / Cs: 2 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature: 85 K / Temperature (max): 90 K / Temperature (min): 80 K
Image recordingElectron dose: 28 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Image scansNum. digital images: 1234
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1CTFFIND3CTF correction
2RELION3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: D8 (2x8 fold dihedral)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11359 / Nominal pixel size: 1.34 Å / Actual pixel size: 1.34 Å
Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Then particle-based beam-induced movement correction ...Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Then particle-based beam-induced movement correction was performed using statistical movie processing in RELION.
Symmetry type: POINT
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms126080 0 432 0 126512

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