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- EMDB-2871: Cryo-EM structure of the Dark apoptosome. -

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Basic information

Entry
Database: EMDB / ID: EMD-2871
TitleCryo-EM structure of the Dark apoptosome.
Map dataReconstruction of Dark apoptosome
Sample
  • Sample: Dark apoptosome
  • Protein or peptide: Dark apoptosome
KeywordsCryo-EM / apoptosome / single particle analysis
Function / homology
Function and homology information


negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / positive regulation of compound eye retinal cell programmed cell death / Formation of apoptosome / salivary gland histolysis / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / central nervous system formation ...negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / positive regulation of compound eye retinal cell programmed cell death / Formation of apoptosome / salivary gland histolysis / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / 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 / dendrite morphogenesis / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / response to gamma radiation / ADP binding / neuron cellular homeostasis / positive regulation of apoptotic process / 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 / 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
Apoptosome protein Dark
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 4.0 Å
AuthorsPang YX / Bai XC / Hao Q / Yan CY / Chen ZQ / Wang JW / Scheres SHW / Shi YG
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
DepositionJan 28, 2015-
Header (metadata) releaseFeb 25, 2015-
Map releaseFeb 25, 2015-
UpdateMar 4, 2015-
Current statusMar 4, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j9l
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j9l
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_2871.png

Downloads & links

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Map

FileDownload / File: emd_2871.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Dark apoptosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.0643 / Movie #1: 0.07
Minimum - Maximum-0.22986917 - 0.40449759
Average (Standard dev.)0.0000037 (±0.02104086)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2300.4040.000

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Supplemental data

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Sample components

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Entire : Dark apoptosome

EntireName: Dark apoptosome
Components
  • Sample: Dark apoptosome
  • Protein or peptide: Dark apoptosome

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Supramolecule #1000: Dark apoptosome

SupramoleculeName: Dark apoptosome / type: sample / ID: 1000 / Oligomeric state: homo 16 mers / Number unique components: 1
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.5 MDa

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Macromolecule #1: Dark apoptosome

MacromoleculeName: Dark apoptosome / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly / Cell: Hi-5 insect cells
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.5 MDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly) / Recombinant cell: Hi-5 insect cells / Recombinant plasmid: pFastBac1

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 8 / Details: 25mM Tris, 150mM NaCl, 5mM dithiothreitol
StainingType: NEGATIVE
Details: Grids were blotted for 2 seconds and flash frozen in liquid ethane using an FEI Vitrobot.
GridDetails: Samples were placed on glow-discharged holey carbon grids (Quantifoil CuR2/2), on which a home-made continuous carbon film had previously been deposited.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK II / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
DateNov 17, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 693 / Average electron dose: 28 e/Å2
Details: 16 video frames were recorded in 1s by FalconII detector.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104748 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 6.6 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: D8 (2x8 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION
Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, 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. Second, particle based beam-induced movement correction was performed using statistical movie processing in RELION.
Number images used: 9354

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