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- EMDB-5235: Structure of the Drosophila apoptosome -

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Basic information

Entry
Database: EMDB / ID: EMD-5235
TitleStructure of the Drosophila apoptosome
Map datamap of the Drosophila apoptosome
Sample
  • Sample: Drosophila apoptosome (double ring)
  • Protein or peptide: Drosophila Apaf-1 like protein
KeywordsDrosophila apoptosome / apoptosis / 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
Apaf-1 related killer DARK
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsYuan S / Yu X / Topf M / Ludtke SJ / Akey CW
CitationJournal: Structure / Year: 2011
Title: Structure of the Drosophila apoptosome at 6.9 å resolution.
Authors: Shujun Yuan / Xinchao Yu / Maya Topf / Loretta Dorstyn / Sharad Kumar / Steven J Ludtke / Christopher W Akey /
Abstract: The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc ...The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.
History
DepositionSep 22, 2010-
Header (metadata) releaseDec 9, 2010-
Map releaseJul 7, 2011-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v4l
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5235_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5235.map.gz / Format: CCP4 / Size: 89 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of the Drosophila apoptosome
Voxel sizeX=Y=Z: 1.72 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-1.28431416 - 3.61963797
Average (Standard dev.)0.0321025 (±0.20919579)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-144-144-144
Dimensions288288288
Spacing288288288
CellA=B=C: 495.36002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.721.721.72
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z495.360495.360495.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-144-144-144
NC/NR/NS288288288
D min/max/mean-1.2843.6200.032

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Supplemental data

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Sample components

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Entire : Drosophila apoptosome (double ring)

EntireName: Drosophila apoptosome (double ring)
Components
  • Sample: Drosophila apoptosome (double ring)
  • Protein or peptide: Drosophila Apaf-1 like protein

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Supramolecule #1000: Drosophila apoptosome (double ring)

SupramoleculeName: Drosophila apoptosome (double ring) / type: sample / ID: 1000
Details: Sample assembled in low salt buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) at about 0.5 mg per ml with dATP
Oligomeric state: hexadecamer of Dark molecules / Number unique components: 1
Molecular weightTheoretical: 2.5 MDa

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Macromolecule #1: Drosophila Apaf-1 like protein

MacromoleculeName: Drosophila Apaf-1 like protein / type: protein_or_peptide / ID: 1 / Name.synonym: Dark / Number of copies: 16 / Oligomeric state: hexadecamer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: fruit fly / Location in cell: cytosol
Molecular weightTheoretical: 160 KDa
Recombinant expressionOrganism: sf21 insect cells (unknown) / Recombinant plasmid: pFastBac

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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

Concentration2 mg/mL
BufferpH: 7.5
Details: 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT
GridDetails: thin carbon film covered holey grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: OTHER
Details: Vitrification instrument: FEI Vitrobot. blotting at room temperature with sample at room temperature
Method: Blot for 2-2.5s before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93 K / Max: 100 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification
Detailsactual magnification at the ccd 87000, camera pixel size 15um, 1.72 angstrom per pixel, data collected semi-automatically with EMTools
DateSep 15, 2009
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 1100 / Average electron dose: 20 e/Å2
Details: the frames were 4096 x 4096 tiffs collected with the TVIPS CCD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final two d classificationNumber classes: 1000
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2
Details: Projection matching was done with Fourier ring correlation, model-based masking and SSNR weighting over an 80-6 angstrom resolution range. The final refinement steps used an angular step of ...Details: Projection matching was done with Fourier ring correlation, model-based masking and SSNR weighting over an 80-6 angstrom resolution range. The final refinement steps used an angular step of 2.5 degrees and each of the 48,000 particles was matched to the best two projection classes (1353). In total, 45,000 particles were used in the final reconstruction and the 3D map was amplitude corrected then Gaussian low-pass filtered with a Fourier half-width of 0.12.
Number images used: 48271
Detailsactual class number 1353

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