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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-1931 | |||||||||
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Title | Map of the apoptosome-procaspase-9 complex | |||||||||
![]() | Map of the apoptosome-procaspase-9 complex | |||||||||
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![]() | apoptosome / Apaf-1 / procaspase-9 activation | |||||||||
Function / homology | Apoptotic protease-activating factor 1![]() | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 16.9 Å | |||||||||
![]() | Yuan S / Ludtke SJ / Akey CW | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 24.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.7 KB 12.7 KB | Display Display | ![]() |
Images | ![]() | 157.6 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 204.4 KB | Display | ![]() |
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Full document | ![]() | 203.4 KB | Display | |
Data in XML | ![]() | 6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map of the apoptosome-procaspase-9 complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.9 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Human apoptosome-procaspase-9 complex
Entire | Name: Human apoptosome-procaspase-9 complex |
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Components |
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-Supramolecule #1000: Human apoptosome-procaspase-9 complex
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 1.2 MDa |
-Macromolecule #1: Apaf-1
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 130 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | InterPro: Apoptotic protease-activating factor 1 |
-Macromolecule #2: Procaspase-9
Macromolecule | Name: Procaspase-9 / type: protein_or_peptide / ID: 2 / Name.synonym: Procaspase-9 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 50 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #3: Cytochrome c
Macromolecule | Name: Cytochrome c / type: protein_or_peptide / ID: 3 / Name.synonym: Cytochrome c / Number of copies: 7 / Oligomeric state: Monomer / Recombinant expression: No |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 10 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 3 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT |
Grid | Details: Quantifoil holey grids |
Vitrification | Cryogen 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
Microscope | FEI TECNAI F20 |
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Temperature | Average: 96 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification |
Details | 4k x 4k ccd used |
Image recording | Category: CCD / Film or detector model: GENERIC TVIPS / Number real images: 447 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 29000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 29000 |
Sample stage | Specimen holder: Side entry liquid nitrogen-cooled cryo specimen holder Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | Apaf-1 was co-assembled with bovine cytochrome c and pc-9 |
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CTF correction | Details: Each image |
Final reconstruction | Applied 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 |
Final two d classification | Number classes: 1000 |
-Atomic model buiding 1
Initial model | PDB ID: ![]() 3iza |
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Software | Name: ![]() |
Details | Protocol: rigid body |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation |
-Atomic model buiding 2
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: ![]() |
Details | PDBEntryID_givenInChain. Protocol: rigid body. final fit was done manually taking into account the orientation of the p20-p10 loop. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |