+
Open data
-
Basic information
Entry | Database: PDB / ID: 3j2t | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | An improved model of the human apoptosome | |||||||||
![]() |
| |||||||||
![]() | APOPTOSIS / Apoptosis protease activating factor-1 / Apaf-1 / cytochrome c | |||||||||
Function / homology | ![]() Release of apoptotic factors from the mitochondria / Formation of apoptosome / Activation of caspases through apoptosome-mediated cleavage / Pyroptosis / Regulation of the apoptosome activity / Transcriptional activation of mitochondrial biogenesis / response to G1 DNA damage checkpoint signaling / activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c / Formation of apoptosome / regulation of apoptotic DNA fragmentation ...Release of apoptotic factors from the mitochondria / Formation of apoptosome / Activation of caspases through apoptosome-mediated cleavage / Pyroptosis / Regulation of the apoptosome activity / Transcriptional activation of mitochondrial biogenesis / response to G1 DNA damage checkpoint signaling / activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c / Formation of apoptosome / regulation of apoptotic DNA fragmentation / Detoxification of Reactive Oxygen Species / apoptosome / TP53 Regulates Metabolic Genes / Cytoprotection by HMOX1 / Respiratory electron transport / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / SMAC (DIABLO) binds to IAPs / SMAC(DIABLO)-mediated dissociation of IAP:caspase complexes / mitochondrial electron transport, cytochrome c to oxygen / mitochondrial electron transport, ubiquinol to cytochrome c / TP53 Regulates Transcription of Caspase Activators and Caspases / Transcriptional Regulation by E2F6 / cysteine-type endopeptidase activator activity involved in apoptotic process / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / : / response to nutrient / forebrain development / cardiac muscle cell apoptotic process / cellular response to transforming growth factor beta stimulus / heat shock protein binding / intrinsic apoptotic signaling pathway / positive regulation of apoptotic signaling pathway / kidney development / neural tube closure / mitochondrial intermembrane space / activation of cysteine-type endopeptidase activity involved in apoptotic process / ADP binding / nervous system development / secretory granule lumen / neuron apoptotic process / regulation of apoptotic process / ficolin-1-rich granule lumen / cell differentiation / response to hypoxia / electron transfer activity / positive regulation of apoptotic process / nucleotide binding / heme binding / Neutrophil degranulation / apoptotic process / protein-containing complex / extracellular exosome / extracellular region / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.5 Å | |||||||||
![]() | Yuan, S. / Topf, M. / Akey, C.W. | |||||||||
![]() | ![]() Title: Changes in Apaf-1 conformation that drive apoptosome assembly. Authors: Shujun Yuan / Maya Topf / Thomas F Reubold / Susanne Eschenburg / Christopher W Akey / ![]() Abstract: Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full ...Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate β-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory β-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by β-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death. #1: ![]() Title: Structure of an apoptosome-procaspase-9 CARD complex. Authors: Shujun Yuan / Xinchao Yu / Maya Topf / Steven J Ludtke / Xiaodong Wang / Christopher W Akey / ![]() Abstract: Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed ...Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at approximately 9.5 A resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem seven and eight blade beta-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly tethered "disk" that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub. | |||||||||
History |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | Molecule: ![]() ![]() |
-
Downloads & links
-
Download
PDBx/mmCIF format | ![]() | 1.5 MB | Display | ![]() |
---|---|---|---|---|
PDB format | ![]() | 1.2 MB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
---|
-Related structure data
Related structure data | ![]() 5186M M: map data used to model this data |
---|---|
Similar structure data |
-
Links
-
Assembly
Deposited unit | ![]()
|
---|---|
1 |
|
-
Components
#1: Protein | Mass: 144095.812 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #2: Protein | Mass: 11595.392 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #3: Chemical | ChemComp-ATP / #4: Chemical | ChemComp-HEM / |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-
Sample preparation
Component |
| |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer solution | Name: low salt HEPES buffer / pH: 7.5 Details: 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT | |||||||||||||||
Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Details: Quantifoil R1.2/1.3 holey grid | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temp: 77 K / Humidity: 100 % Details: Blot for 2 seconds before plunging (FEI VITROBOT MARK III) Method: Blot for 2 seconds before plunging |
-
Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TECNAI F20 / Date: Aug 1, 2008 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 62000 X / Calibrated magnification: 62000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm |
Specimen holder | Specimen holder model: GATAN LIQUID NITROGEN Specimen holder type: Side entry liquid nitrogen-cooled cryo specimen holder Temperature: 96 K / Tilt angle max: 0 ° / Tilt angle min: 0 ° |
Image recording | Electron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM |
Image scans | Num. digital images: 400 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Relative weight: 1 |
-
Processing
EM software |
| |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTF correction | Details: CTF correction was done on each particle image based on summed power spectra from each micrograph. | |||||||||||||||
Symmetry | Point symmetry: C7 (7 fold cyclic) | |||||||||||||||
3D reconstruction | Method: Projection Matching / Resolution: 9.5 Å / Num. of particles: 34000 / Nominal pixel size: 2.26 Å / Actual pixel size: 2.26 Å / Magnification calibration: TMV images Details: EMAN was used for reconstruction using C7 symmetry. Symmetry type: POINT | |||||||||||||||
Atomic model building |
| |||||||||||||||
Atomic model building |
| |||||||||||||||
Refinement step | Cycle: LAST
|