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- PDB-5juy: Active human apoptosome with procaspase-9 -

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Basic information

Entry
Database: PDB / ID: 5juy
TitleActive human apoptosome with procaspase-9
Components
  • Apoptotic protease-activating factor 1
  • Caspase-9
  • Cytochrome c
KeywordsAPOPTOSIS / Apaf-1 / programmed cell death / AAA+ ATPase
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 / caspase-9 / Transcriptional activation of mitochondrial biogenesis / response to G1 DNA damage checkpoint signaling / caspase complex / : ...Release of apoptotic factors from the mitochondria / Formation of apoptosome / Activation of caspases through apoptosome-mediated cleavage / Pyroptosis / Regulation of the apoptosome activity / caspase-9 / Transcriptional activation of mitochondrial biogenesis / response to G1 DNA damage checkpoint signaling / caspase complex / : / regulation of apoptotic DNA fragmentation / Formation of apoptosome / Detoxification of Reactive Oxygen Species / apoptosome / TP53 Regulates Metabolic Genes / Cytoprotection by HMOX1 / leukocyte apoptotic process / glial cell apoptotic process / response to cobalt ion / : / Respiratory electron transport / Caspase activation via Dependence Receptors in the absence of ligand / Activation of caspases through apoptosome-mediated cleavage / fibroblast apoptotic process / Regulation of the apoptosome activity / SMAC (DIABLO) binds to IAPs / SMAC(DIABLO)-mediated dissociation of IAP:caspase complexes / : / AKT phosphorylates targets in the cytosol / mitochondrial electron transport, cytochrome c to oxygen / epithelial cell apoptotic process / platelet formation / mitochondrial electron transport, ubiquinol to cytochrome c / TP53 Regulates Transcription of Caspase Activators and Caspases / Transcriptional Regulation by E2F6 / Constitutive Signaling by AKT1 E17K in Cancer / cysteine-type endopeptidase activator activity involved in apoptotic process / protein maturation / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / signal transduction in response to DNA damage / : / cellular response to transforming growth factor beta stimulus / forebrain development / heat shock protein binding / cardiac muscle cell apoptotic process / enzyme activator activity / intrinsic apoptotic signaling pathway / cellular response to dexamethasone stimulus / response to nutrient / kidney development / neural tube closure / response to ischemia / positive regulation of apoptotic signaling pathway / ADP binding / NOD1/2 Signaling Pathway / : / mitochondrial intermembrane space / protein processing / SH3 domain binding / intrinsic apoptotic signaling pathway in response to DNA damage / positive regulation of neuron apoptotic process / cellular response to UV / response to estradiol / peptidase activity / nervous system development / secretory granule lumen / regulation of apoptotic process / neuron apoptotic process / ficolin-1-rich granule lumen / response to lipopolysaccharide / electron transfer activity / cell differentiation / response to hypoxia / positive regulation of apoptotic process / cysteine-type endopeptidase activity / nucleotide binding / DNA damage response / heme binding / Neutrophil degranulation / apoptotic process / protein kinase binding / protein-containing complex / mitochondrion / extracellular exosome / extracellular region / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
CASP9, CARD domain / 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 / Caspase recruitment domain / Apoptotic protease-activating factors, helical domain / NB-ARC ...CASP9, CARD domain / 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 / Caspase recruitment 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 / Peptidase family C14A, His active site / Caspase family histidine active site. / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. / Caspase family p10 domain profile. / Peptidase C14A, caspase catalytic domain / Caspase, interleukin-1 beta converting enzyme (ICE) homologues / Peptidase C14, p20 domain / Caspase family p20 domain profile. / : / Caspase domain / Caspase-like domain superfamily / 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. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / HEME C / Apoptotic protease-activating factor 1 / Caspase-9 / Cytochrome c
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsCheng, T.C. / Hong, C. / Akey, I.V. / Yuan, S. / Akey, C.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM63834 United States
CitationJournal: Elife / Year: 2016
Title: A near atomic structure of the active human apoptosome.
Authors: Tat Cheung Cheng / Chuan Hong / Ildikó V Akey / Shujun Yuan / Christopher W Akey /
Abstract: In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo- ...In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.
History
DepositionMay 10, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 19, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2016Group: Other
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.4Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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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
O: Caspase-9
P: Caspase-9
Q: Caspase-9
R: Caspase-9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,127,66432
Polymers1,119,89618
Non-polymers7,76814
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 142023.672 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Details: without N-terminal CARDs / Source: (gene. exp.) Homo sapiens (human) / Gene: APAF1, KIAA0413 / Production host: Spodoptera frugiperda (fall armyworm) / 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: Protein
Caspase-9 / CASP-9 / Apoptotic protease Mch-6 / Apoptotic protease-activating factor 3 / APAF-3 / ICE-like ...CASP-9 / Apoptotic protease Mch-6 / Apoptotic protease-activating factor 3 / APAF-3 / ICE-like apoptotic protease 6 / ICE-LAP6


Mass: 11140.723 Da / Num. of mol.: 4 / Fragment: N-terminal caspase recognition domain
Source method: isolated from a genetically manipulated source
Details: N-terminal caspase recognition domain (CARD) of procaspase-9
Source: (gene. exp.) Homo sapiens (human) / Gene: CASP9, MCH6
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P55211, caspase-9
#4: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O12P3
#5: Chemical
ChemComp-HEC / HEME C


Mass: 618.503 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C34H34FeN4O4
Has protein modificationY

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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

ComponentName: Active complex of Apaf-1, cytochrome c and pro-caspase 9
Type: COMPLEX
Details: Complex stoichiometry: 4 full length Apaf-1 molecules with ordered N-terminal CARDs, 3 truncated Apaf-1 molecules without CARDs, 7 cytochrome c molecules and 4 N-terminal CARDs from procaspase-9
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 1.3 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: pFast-Bac
Buffer solutionpH: 7.5 / Details: Buffer A
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepes1
220 mMpotassium chlorideKCL1
31 mMEDTA1
41 mMEGTA1
51.5 mMmagnesium chlorideMg2Cl21
61.0 mMdATP1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: data collected with a Cs aberration corrector and a GIF energy filter with a slit width of 20 eV.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1500 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 1.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1991
EM imaging opticsEnergyfilter name: GIF
Image scansWidth: 7676 / Height: 7420 / Movie frames/image: 23 / Used frames/image: 2-23

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Processing

EM software
IDNameVersionCategory
1RELION1.3particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7MDFFmodel fitting
9RELION1.3initial Euler assignment
10RELION1.3final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 134970
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92867 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial rigid body fitting with Chimera with a previously published starting model (PDB 3J2T), molecular dynamics flexible fitting with MDFF and real space density refinement with Phenix

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