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- PDB-3j2t: An improved model of the human apoptosome -

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Basic information

Entry
Database: PDB / ID: 3j2t
TitleAn improved model of the human apoptosome
Components
  • Apoptotic protease-activating factor 1
  • Cytochrome c
KeywordsAPOPTOSIS / Apoptosis protease activating factor-1 / Apaf-1 / cytochrome c
Function / homology
Function and homology information


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 / Detoxification of Reactive Oxygen Species / regulation of apoptotic DNA fragmentation / Formation of apoptosome ...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 / Detoxification of Reactive Oxygen Species / regulation of apoptotic DNA fragmentation / Formation of apoptosome / apoptosome / activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c / TP53 Regulates Metabolic Genes / Cytoprotection by HMOX1 / Respiratory electron transport / Regulation of the apoptosome activity / Activation of caspases through apoptosome-mediated cleavage / 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 / respirasome / forebrain development / positive regulation of apoptotic signaling pathway / cardiac muscle cell apoptotic process / cellular response to transforming growth factor beta stimulus / heat shock protein binding / intrinsic apoptotic signaling pathway / response to nutrient / kidney development / neural tube closure / ADP binding / mitochondrial intermembrane space / activation of cysteine-type endopeptidase activity involved in apoptotic process / nervous system development / neuron apoptotic process / regulation of apoptotic process / secretory granule lumen / ficolin-1-rich granule lumen / electron transfer activity / cell differentiation / response to hypoxia / positive regulation of apoptotic process / nucleotide binding / apoptotic process / heme binding / Neutrophil degranulation / protein-containing complex / extracellular exosome / extracellular region / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol
Similarity search - Function
Apoptotic Protease-Activating Factor 1, CARD domain / : / Apoptotic protease-activating factor 1-like, winged-helix domain / Apoptotic protease-activating factor 1 / APAF-1 helical domain / APAF-1 helical domain / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / Cytochrome c, class IA/ IB ...Apoptotic Protease-Activating Factor 1, CARD domain / : / Apoptotic protease-activating factor 1-like, winged-helix domain / Apoptotic protease-activating factor 1 / APAF-1 helical domain / APAF-1 helical domain / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / Cytochrome c, class IA/ IB / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Cytochrome c / Cytochrome c family profile. / Cytochrome c-like domain / Cytochrome c-like domain superfamily / Death-like domain superfamily / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / 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
ADENOSINE-5'-TRIPHOSPHATE / PROTOPORPHYRIN IX CONTAINING FE / Apoptotic protease-activating factor 1 / Cytochrome c
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.5 Å
AuthorsYuan, S. / Topf, M. / Akey, C.W.
Citation
Journal: Biochemistry / Year: 2013
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: Journal: Structure / Year: 2010
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
DepositionDec 23, 2012Deposition site: RCSB / Processing site: RCSB
SupersessionApr 10, 2013ID: 3IZA
Revision 1.0Apr 10, 2013Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2013Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id

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Structure visualization

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Assembly

Deposited unit
A: Apoptotic protease-activating factor 1
B: Apoptotic protease-activating factor 1
C: Apoptotic protease-activating factor 1
D: Apoptotic protease-activating factor 1
E: Apoptotic protease-activating factor 1
F: Apoptotic protease-activating factor 1
G: Apoptotic protease-activating factor 1
H: Cytochrome c
I: Cytochrome c
J: Cytochrome c
K: Cytochrome c
L: Cytochrome c
M: Cytochrome c
N: Cytochrome c
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,097,70428
Polymers1,089,83814
Non-polymers7,86614
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Apoptotic protease-activating factor 1 / APAF-1


Mass: 144095.812 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: Apaf-1, APAF1, KIAA0413 / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): sf21 / References: UniProt: O14727
#2: Protein
Cytochrome c /


Mass: 11595.392 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P62894
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C34H32FeN4O4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent-ID
1Human Apaf-1 apoptosome with stably bound cytochrome c and procaspase-9 CARDs.COMPLEXRecombinant procaspase-9 was added to form pc-9 apoptosomes and thrombin was added to cleave an engineered site in the CARD-p20 linker of pc-9. This released catalytic domains of pc-9 and left pc-9 CARDs bound to flexibly attached Apaf-1 CARDs which together form a flexibly attached disk that sits on top of the Apaf-1 platform. The CARD-CARD disk has not been modeled due to flexibility.0
2Apaf-1APAF17 Apaf-1 and 7 cytochrome c molecules were co-assembled to form a heptameric apoptosome1
Buffer solutionName: low salt HEPES buffer / pH: 7.5
Details: 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil R1.2/1.3 holey grid
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Aug 1, 2008
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 62000 X / Calibrated magnification: 62000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Specimen holderSpecimen 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 recordingElectron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 400
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Rosettamodel fitting
2Situsmodel fitting
3UCSF Chimeramodel fitting
4EMAN3D reconstruction
CTF correctionDetails: CTF correction was done on each particle image based on summed power spectra from each micrograph.
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionMethod: 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
IDProtocolSpaceTarget criteriaDetails
1FLEXIBLE FITREALCross-correlation coefficientMETHOD--flexible fitting REFINEMENT PROTOCOL--Rigid body docking followed by flexible fitting DETAILS--Modeller was used to create a full length human Apaf-1. Initial fit was done using Chimera rigid body fitting. Rosetta was used for flexible fitting.
2RIGID BODY FITREALCross-correlation coefficientMETHOD--rigid body fitting REFINEMENT PROTOCOL--rigid body DETAILS--Cytochrome c was fitted into its local density using Situs.
Atomic model building
IDPDB-ID 3D fitting-ID
11Z6T

1z6t

1
23SFZ

3sfz

1
32B4Z

2b4z

2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms69671 0 518 0 70189

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