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- PDB-3j9l: Structure of Dark apoptosome from Drosophila melanogaster -

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Basic information

Entry
Database: PDB / ID: 3j9l
TitleStructure of Dark apoptosome from Drosophila melanogaster
ComponentsApaf-1 related killer DARK
KeywordsAPOPTOSIS / programmed cell death
Function / homology
Function and homology information


negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / Regulation of the apoptosome activity / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / central nervous system formation / : ...negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / Regulation of the apoptosome activity / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / central nervous system formation / : / chaeta development / sperm individualization / apoptosome / S-adenosylmethionine cycle / Neutrophil degranulation / CARD domain binding / programmed cell death / triglyceride homeostasis / dendrite morphogenesis / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / ADP binding / response to gamma radiation / neuron cellular homeostasis / positive regulation of apoptotic process / ATP binding / identical protein binding
Similarity search - Function
APAF-1 helical domain / APAF-1 helical domain / NB-ARC / NB-ARC domain / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Apaf-1 related killer DARK
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 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
C: Apaf-1 related killer DARK
D: Apaf-1 related killer DARK
E: Apaf-1 related killer DARK
F: Apaf-1 related killer DARK
G: Apaf-1 related killer DARK
H: Apaf-1 related killer DARK
I: Apaf-1 related killer DARK
J: Apaf-1 related killer DARK
K: Apaf-1 related killer DARK
L: Apaf-1 related killer DARK
M: Apaf-1 related killer DARK
N: Apaf-1 related killer DARK
O: Apaf-1 related killer DARK
P: Apaf-1 related killer DARK
Q: Apaf-1 related killer DARK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,812,71631
Polymers1,805,34816
Non-polymers7,36815
Water0
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: 112834.266 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Source: (natural) Drosophila melanogaster (fruit fly) / References: UniProt: Q7KLI1
#2: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C10H16N5O12P3

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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: Dark apoptosome / Type: COMPLEX
Molecular weightValue: 2.5 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: Nov 17, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 78000 X / Calibrated magnification: 104748 X / Nominal defocus max: 6600 nm / Nominal defocus min: 1600 nm / Cs: 2 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / 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: 693
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 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9354 / 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. atoms112501 0 450 0 112951

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