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- EMDB-1931: Map of the apoptosome-procaspase-9 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-1931
TitleMap of the apoptosome-procaspase-9 complex
Map dataMap of the apoptosome-procaspase-9 complex
Sample
  • Sample: Human apoptosome-procaspase-9 complex
  • Protein or peptide: Apaf-1APAF1
  • Protein or peptide: Procaspase-9
  • Protein or peptide: Cytochrome c
Keywordsapoptosome / Apaf-1 / procaspase-9 activation
Function / homologyApoptotic protease-activating factor 1
Function and homology information
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.9 Å
AuthorsYuan S / Ludtke SJ / Akey CW
CitationJournal: Structure / Year: 2011
Title: The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3.
Authors: Shujun Yuan / Xinchao Yu / John M Asara / John E Heuser / Steven J Ludtke / Christopher W Akey /
Abstract: Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD- ...Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.
History
DepositionJul 12, 2011-
Header (metadata) releaseMar 15, 2012-
Map releaseMar 15, 2012-
UpdateMar 15, 2012-
Current statusMar 15, 2012Processing site: PDBe / 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 cylindrical radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image1931.png

Downloads & links

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Map

FileDownload / File: emd_1931.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the apoptosome-procaspase-9 complex
Voxel sizeX=Y=Z: 2.9 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-0.67261672 - 2.2649889
Average (Standard dev.)0.01628205 (±0.14226763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 556.80005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z556.800556.800556.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.6732.2650.016

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Supplemental data

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Sample components

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Entire : Human apoptosome-procaspase-9 complex

EntireName: Human apoptosome-procaspase-9 complex
Components
  • Sample: Human apoptosome-procaspase-9 complex
  • Protein or peptide: Apaf-1APAF1
  • Protein or peptide: Procaspase-9
  • Protein or peptide: Cytochrome c

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Supramolecule #1000: Human apoptosome-procaspase-9 complex

SupramoleculeName: Human apoptosome-procaspase-9 complex / type: sample / ID: 1000
Details: A slight excess of procaspase-9 was added to ensure saturation of binding to sites on the apoptosome.
Oligomeric state: 5-7 procaspase-9 molecules bound to the human apoptosome comprised of 7 Apaf-1 subunits
Number unique components: 3
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: Apaf-1

MacromoleculeName: Apaf-1 / type: protein_or_peptide / ID: 1 / Name.synonym: Apaf-1
Details: Apaf-1, cytochrome c and procaspase-9 were co-assembled in the presence of dATP to form the apoptosome-procaspase-9 complex
Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytosol
Molecular weightTheoretical: 130 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac1
SequenceInterPro: Apoptotic protease-activating factor 1

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Macromolecule #2: Procaspase-9

MacromoleculeName: Procaspase-9 / type: protein_or_peptide / ID: 2 / Name.synonym: Procaspase-9 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytosol
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21

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Macromolecule #3: Cytochrome c

MacromoleculeName: Cytochrome c / type: protein_or_peptide / ID: 3 / Name.synonym: Cytochrome c / Number of copies: 7 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Cow / Tissue: heart / Location in cell: Mitochondria
Molecular weightTheoretical: 10 KDa

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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

Concentration3 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT
GridDetails: Quantifoil holey grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III / Details: Vitrification instrument: Vitrobot Mark 3 (FEI) / Method: Blot for 2-3 seconds before plunging at 20C

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 29000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 29000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 96 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification
Details4k x 4k ccd used
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS / Number real images: 447 / Average electron dose: 20 e/Å2
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2
Details: 3D volume was calculated without imposing any symmetry (c1)
Number images used: 20000
DetailsApaf-1 was co-assembled with bovine cytochrome c and pc-9

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Atomic model buiding 1

Initial modelPDB ID:

3iza
PDB Unreleased entry

SoftwareName: chimera
DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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Atomic model buiding 2

Initial modelPDB ID:

1jxq


Chain - Chain ID: A
SoftwareName: chimera
DetailsPDBEntryID_givenInChain. Protocol: rigid body. final fit was done manually taking into account the orientation of the p20-p10 loop.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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