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- EMDB-6690: Apaf-1-Caspase-9 holoenzyme -

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Basic information

Entry
Database: EMDB / ID: EMD-6690
TitleApaf-1-Caspase-9 holoenzyme
Map dataApaf-1-Caspase-9 holoenzyme platform
Sample
  • Complex: Apaf-1 apoptosome
    • Complex: Apaf-1
      • Protein or peptide: Apoptotic protease-activating factor 1
    • Complex: cytochrome c
      • Protein or peptide: Cytochrome c
    • Complex: Apaf-1 CARD
      • Protein or peptide: Apoptotic protease-activating factor 1
    • Complex: caspase-9 CARD
      • Protein or peptide: Caspase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
Keywordsapoptosis holoenzyme / APOPTOSIS
Function / homology
Function and homology information


caspase-9 / response to G1 DNA damage checkpoint signaling / caspase complex / regulation of apoptotic DNA fragmentation / cytochrome c-heme linkage / Formation of apoptosome / apoptosome / cytochrome complex / leukocyte apoptotic process / glial cell apoptotic process ...caspase-9 / response to G1 DNA damage checkpoint signaling / caspase complex / regulation of apoptotic DNA fragmentation / cytochrome c-heme linkage / Formation of apoptosome / apoptosome / cytochrome complex / leukocyte apoptotic process / glial cell apoptotic process / cysteine-type endopeptidase activator activity / response to cobalt ion / Caspase activation via Dependence Receptors in the absence of ligand / 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 / fibroblast apoptotic process / AKT phosphorylates targets in the cytosol / mitochondrial electron transport, cytochrome c to oxygen / epithelial cell apoptotic process / cysteine-type endopeptidase activator activity involved in apoptotic process / platelet formation / response to anesthetic / mitochondrial electron transport, ubiquinol to cytochrome c / TP53 Regulates Transcription of Caspase Activators and Caspases / Constitutive Signaling by AKT1 E17K in Cancer / Transcriptional Regulation by E2F6 / positive regulation of execution phase of apoptosis / 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 / response to nutrient / cellular response to dexamethasone stimulus / intrinsic apoptotic signaling pathway / protein maturation / positive regulation of apoptotic signaling pathway / response to ischemia / NOD1/2 Signaling Pathway / neural tube closure / apoptotic signaling pathway / kidney development / enzyme activator activity / ADP binding / protein processing / SH3 domain binding / mitochondrial intermembrane space / intrinsic apoptotic signaling pathway in response to DNA damage / cellular response to UV / nervous system development / response to estradiol / peptidase activity / positive regulation of neuron apoptotic process / secretory granule lumen / regulation of apoptotic process / neuron apoptotic process / response to lipopolysaccharide / ficolin-1-rich granule lumen / cell differentiation / response to hypoxia / electron transfer activity / positive regulation of apoptotic process / cysteine-type endopeptidase activity / nucleotide binding / apoptotic process / lipid binding / heme binding / DNA damage response / Neutrophil degranulation / protein kinase binding / protein-containing complex / mitochondrion / proteolysis / extracellular exosome / extracellular region / ATP binding / metal ion binding / identical protein binding / nucleus / 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 / Apoptotic protease-activating factors, helical domain / Caspase recruitment 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 / Apoptotic protease-activating factors, helical domain / Caspase recruitment domain / NB-ARC / NB-ARC domain / Cytochrome c, class IA/ IB / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Peptidase C14 family / 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 / Cytochrome c / Caspase-like domain superfamily / Cytochrome c family profile. / Cytochrome c-like domain / Cytochrome c-like domain superfamily / Death-like domain superfamily / WD domain, G-beta repeat / 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. / 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
cDNA FLJ75893, highly similar to Homo sapiens caspase 9, apoptosis-related cysteine peptidase (CASP9), transcript variant alpha, mRNA / Apoptotic protease-activating factor 1 / Cytochrome c / Caspase-9
Similarity search - Component
Biological speciesHomo sapiens (human) / Equus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsLi Y / Zhou M
Funding support China, United Kingdom, 3 items
OrganizationGrant numberCountry
Ministry of Science and Technology2014ZX09507003006 to YS China
National Natural Science Foundation of China(projects 31430020, 31130002, and 31321062 to YS China
UK Medical Research Council(MC_UP_A025_1013, to SHWS United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme.
Authors: Yini Li / Mengying Zhou / Qi Hu / Xiao-Chen Bai / Weiyun Huang / Sjors H W Scheres / Yigong Shi /
Abstract: Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is ...Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.
History
DepositionDec 24, 2016-
Header (metadata) releaseFeb 8, 2017-
Map releaseFeb 8, 2017-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wve
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6690.png

Downloads & links

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Map

FileDownload / File: emd_6690.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApaf-1-Caspase-9 holoenzyme platform
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.09729642 - 0.32490632
Average (Standard dev.)0.00011596797 (±0.011304457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0970.3250.000

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Supplemental data

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Sample components

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Entire : Apaf-1 apoptosome

EntireName: Apaf-1 apoptosome
Components
  • Complex: Apaf-1 apoptosome
    • Complex: Apaf-1
      • Protein or peptide: Apoptotic protease-activating factor 1
    • Complex: cytochrome c
      • Protein or peptide: Cytochrome c
    • Complex: Apaf-1 CARD
      • Protein or peptide: Apoptotic protease-activating factor 1
    • Complex: caspase-9 CARD
      • Protein or peptide: Caspase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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Supramolecule #1: Apaf-1 apoptosome

SupramoleculeName: Apaf-1 apoptosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Apaf-1

SupramoleculeName: Apaf-1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: cytochrome c

SupramoleculeName: cytochrome c / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Supramolecule #4: Apaf-1 CARD

SupramoleculeName: Apaf-1 CARD / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Supramolecule #5: caspase-9 CARD

SupramoleculeName: caspase-9 CARD / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4

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Macromolecule #1: Apoptotic protease-activating factor 1

MacromoleculeName: Apoptotic protease-activating factor 1 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 142.023672 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MDAKARNCLL QHREALEKDI KTSYIMDHMI SDGFLTISEE EKVRNEPTQQ QRAAMLIKMI LKKDNDSYVS FYNALLHEGY KDLAALLHD GIPVVSSSSG KDSVSGITSY VRTVLCEGGV PQRPVVFVTR KKLVNAIQQK LSKLKGEPGW VTIHGMAGCG K SVLAAEAV ...String:
MDAKARNCLL QHREALEKDI KTSYIMDHMI SDGFLTISEE EKVRNEPTQQ QRAAMLIKMI LKKDNDSYVS FYNALLHEGY KDLAALLHD GIPVVSSSSG KDSVSGITSY VRTVLCEGGV PQRPVVFVTR KKLVNAIQQK LSKLKGEPGW VTIHGMAGCG K SVLAAEAV RDHSLLEGCF PGGVHWVSVG KQDKSGLLMK LQNLCTRLDQ DESFSQRLPL NIEEAKDRLR ILMLRKHPRS LL ILDDVWD SWVLKAFDSQ CQILLTTRDK SVTDSVMGPK YVVPVESSLG KEKGLEILSL FVNMKKADLP EQAHSIIKEC KGS PLVVSL IGALLRDFPN RWEYYLKQLQ NKQFKRIRKS SSYDYEALDE AMSISVEMLR EDIKDYYTDL SILQKDVKVP TKVL CILWD METEEVEDIL QEFVNKSLLF CDRNGKSFRY YLHDLQVDFL TEKNCSQLQD LHKKIITQFQ RYHQPHTLSP DQEDC MYWY NFLAYHMASA KMHKELCALM FSLDWIKAKT ELVGPAHLIH EFVEYRHILD EKDCAVSENF QEFLSLNGHL LGRQPF PNI VQLGLCEPET SEVYQQAKLQ AKQEVDNGML YLEWINKKNI TNLSRLVVRP HTDAVYHACF SEDGQRIASC GADKTLQ VF KAETGEKLLE IKAHEDEVLC CAFSTDDRFI ATCSVDKKVK IWNSMTGELV HTYDEHSEQV NCCHFTNSSH HLLLATGS S DCFLKLWDLN QKECRNTMFG HTNSVNHCRF SPDDKLLASC SADGTLKLWD ATSANERKSI NVKQFFLNLE DPQEDMEVI VKCCSWSADG ARIMVAAKNK IFLFDIHTSG LLGEIHTGHH STIQYCDFSP QNHLAVVALS QYCVELWNTD SRSKVADCRG HLSWVHGVM FSPDGSSFLT SSDDQTIRLW ETKKVCKNSA VMLKQEVDVV FQENEVMVLA VDHIRRLQLI NGRTGQIDYL T EAQVSCCC LSPHLQYIAF GDENGAIEIL ELVNNRIFQS RFQHKKTVWH IQFTADEKTL ISSSDDAEIQ VWNWQLDKCI FL RGHQETV KDFRLLKNSR LLSWSFDGTV KVWNIITGNK EKDFVCHQGT VLSCDISHDA TKFSSTSADK TAKIWSFDLL LPL HELRGH NGCVRCSAFS VDSTLLATGD DNGEIRIWNV SNGELLHLCA PLSEEGAATH GGWVTDLCFS PDGKMLISAG GYIK WWNVV TGESSQTFYT NGTNLKKIHV SPDFKTYVTV DNLGILYILQ TLE

UniProtKB: Apoptotic protease-activating factor 1

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Macromolecule #2: Cytochrome c

MacromoleculeName: Cytochrome c / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Equus caballus (horse)
Molecular weightTheoretical: 11.856793 KDa
Recombinant expressionOrganism: Bacteria (eubacteria)
SequenceString:
MGDVEKGKKI FVQKCAQCHT VEKGGKHKTG PNLHGLFGRK TGQAPGFTYT DANKNKGITW KEETLMEYLE NPKKYIPGTK MIFAGIKKK TEREDLIAYL KKATNE

UniProtKB: Cytochrome c

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Macromolecule #3: Apoptotic protease-activating factor 1

MacromoleculeName: Apoptotic protease-activating factor 1 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.575185 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString:
MDAKARNCLL QHREALEKDI KTSYIMDHMI SDGFLTISEE EKVRNEPTQQ QRAAMLIKMI LKKDNDSYVS FYNALLHEGY KDLAALLHD GIPVVSSSSG KDS

UniProtKB: Apoptotic protease-activating factor 1

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Macromolecule #4: Caspase

MacromoleculeName: Caspase / type: protein_or_peptide / ID: 4 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.698449 KDa
Recombinant expressionOrganism: Bacteria (eubacteria)
SequenceString:
MDEADRRLLR RCRLRLVEEL QVDQLWDVLL SRELFRPHMI EDIQRAGSGS RRDQARQLII DLETRGSQAL PLFISCLEDT GQDMLASFL RTNRQAAKLS K

UniProtKB: cDNA FLJ75893, highly similar to Homo sapiens caspase 9, apoptosis-related cysteine peptidase (CASP9), transcript variant alpha, mRNA

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Macromolecule #5: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 7 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 7 / Number of copies: 7 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 240130
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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