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- EMDB-8178: Active human apoptosome with procaspase-9 -

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Basic information

Entry
Database: EMDB / ID: EMD-8178
TitleActive human apoptosome with procaspase-9
Map dataActive human apoptosome with procaspase-9
Sample
  • Complex: Active complex of Apaf-1, cytochrome c and pro-caspase 9
    • Protein or peptide: Apoptotic protease-activating factor 1
    • Protein or peptide: Cytochrome c
    • Protein or peptide: Caspase-9
    • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: HEME C
KeywordsApaf-1 / apoptosis / 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 / Transcriptional activation of mitochondrial biogenesis / caspase-9 / response to G1 DNA damage checkpoint signaling / caspase complex / 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 / caspase-9 / 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 / leukocyte apoptotic process / Cytoprotection by HMOX1 / glial cell apoptotic process / cysteine-type endopeptidase activator activity / response to cobalt ion / Respiratory electron transport / 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 / forebrain development / signal transduction in response to DNA damage / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / positive regulation of execution phase of apoptosis / cellular response to dexamethasone stimulus / cellular response to transforming growth factor beta stimulus / heat shock protein binding / cardiac muscle cell apoptotic process / response to nutrient / intrinsic apoptotic signaling pathway / protein maturation / response to ischemia / positive regulation of apoptotic signaling pathway / neural tube closure / kidney development / enzyme activator activity / NOD1/2 Signaling Pathway / ADP binding / protein processing / SH3 domain binding / mitochondrial intermembrane space / intrinsic apoptotic signaling pathway in response to DNA damage / cellular response to UV / response to estradiol / nervous system development / peptidase activity / positive regulation of neuron apoptotic process / neuron apoptotic process / secretory granule lumen / regulation of 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 / heme binding / DNA damage response / Neutrophil degranulation / protein kinase binding / protein-containing complex / mitochondrion / extracellular exosome / extracellular region / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
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 / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Apoptotic protease-activating factor 1 / Caspase-9 / Cytochrome c
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (domestic cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsCheng TC / Hong C / Akey IV / Yuan S / Akey CW
Funding support United States, 1 items
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
DepositionJun 20, 2016-
Header (metadata) releaseOct 19, 2016-
Map releaseOct 19, 2016-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5juy
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8178.png

Downloads & links

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Map

FileDownload / File: emd_8178.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationActive human apoptosome with procaspase-9
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 320 pix.
= 437.76 Å		1.37 Å/pix.
x 320 pix.
= 437.76 Å		1.37 Å/pix.
x 320 pix.
= 437.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.368 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.08437762 - 0.1979464
Average (Standard dev.)0.00053622416 (±0.0042053256)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 437.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3681.3681.368
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z437.760437.760437.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0840.1980.001

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Supplemental data

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Additional map: Active human apoptosome with procaspase-9

Fileemd_8178_additional.map
AnnotationActive human apoptosome with procaspase-9
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Active complex of Apaf-1, cytochrome c and pro-caspase 9

EntireName: Active complex of Apaf-1, cytochrome c and pro-caspase 9
Components
  • Complex: Active complex of Apaf-1, cytochrome c and pro-caspase 9
    • Protein or peptide: Apoptotic protease-activating factor 1
    • Protein or peptide: Cytochrome c
    • Protein or peptide: Caspase-9
    • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: HEME C

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Supramolecule #1: Active complex of Apaf-1, cytochrome c and pro-caspase 9

SupramoleculeName: Active complex of Apaf-1, cytochrome c and pro-caspase 9
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.3 MDa

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Macromolecule #1: Apoptotic protease-activating factor 1

MacromoleculeName: Apoptotic protease-activating factor 1 / type: protein_or_peptide / ID: 1 / Details: without N-terminal CARDs / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 142.023672 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
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: Bos taurus (domestic cattle)
Molecular weightTheoretical: 11.595392 KDa
SequenceString:
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKG EREDLIAYLK KATNE

UniProtKB: Cytochrome c

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Macromolecule #3: Caspase-9

MacromoleculeName: Caspase-9 / type: protein_or_peptide / ID: 3
Details: N-terminal caspase recognition domain (CARD) of procaspase-9
Number of copies: 4 / Enantiomer: LEVO / EC number: caspase-9
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.140723 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MDEADRRLLR RCRLRLVEEL QVDQLWDALL SRELFRPHMI EDIQRAGSGS RRDQARQLII DLETRGSQAL PLFISCLEDT GQDMLASFL RTNRQA

UniProtKB: Caspase-9

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Macromolecule #4: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 4 / 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 #5: HEME C

MacromoleculeName: HEME C / type: ligand / ID: 5 / Number of copies: 7 / Formula: HEC
Molecular weightTheoretical: 618.503 Da
Chemical component information

ChemComp-HEC:
HEME C

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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

Concentration4.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHepes
20.0 mMpotassium chlorideKCL
1.0 mMEDTA
1.0 mMEGTA
1.5 mMmagnesium chlorideMg2Cl2
1.0 mMdATP

Details: Buffer A
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF
Detailsdata collected with a Cs aberration corrector and a GIF energy filter with a slit width of 20 eV.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 2-23 / Number grids imaged: 1 / Number real images: 1991 / Average exposure time: 0.3 sec. / Average electron dose: 1.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 134970
Startup modelType of model: EMDB MAP
EMDB ID:

1931


Details: low pass filtered to 60 angstroms
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 92867
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)

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Atomic model buiding 1

DetailsInitial 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
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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