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- EMDB-5186: Structure of an apoptosome-procaspase-9 CARD complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5186
TitleStructure of an apoptosome-procaspase-9 CARD complex
Map dataStructure of the human apoptosome with procaspase-9 CARD
Sample
  • Sample: human apoptosome with bound procaspase-9 CARD
  • Protein or peptide: Apaf-1
  • Protein or peptide: procaspase-9
  • Protein or peptide: cytochrome c
Keywordsapoptosome / Apaf-1 / procaspase-9 CARD / apoptosis
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 / : / 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 / : / regulation of apoptotic DNA fragmentation / Formation of apoptosome / 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 / : / cellular response to transforming growth factor beta stimulus / forebrain development / heat shock protein binding / cardiac muscle cell apoptotic process / intrinsic apoptotic signaling pathway / response to nutrient / kidney development / neural tube closure / positive regulation of apoptotic signaling pathway / ADP binding / : / mitochondrial intermembrane space / nervous system development / secretory granule lumen / regulation of apoptotic process / neuron apoptotic process / ficolin-1-rich granule lumen / electron transfer activity / cell differentiation / response to hypoxia / 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
Apoptotic Protease-Activating Factor 1, CARD domain / : / Apoptotic protease-activating factor 1-like, winged-helix domain / Apoptotic protease-activating factor 1 / Apoptotic protease-activating factor 1 / APAF-1 helical domain / APAF-1 helical domain / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain ...Apoptotic Protease-Activating Factor 1, CARD domain / : / Apoptotic protease-activating factor 1-like, winged-helix domain / Apoptotic protease-activating factor 1 / 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. / 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
Apoptotic protease-activating factor 1 / Cytochrome c
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.5 Å
AuthorsYuan S / Yu X / Topf M / Ludtke SJ / Wang X / Akey CW
CitationJournal: 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
DepositionApr 13, 2010-
Header (metadata) releaseApr 29, 2010-
Map releaseMay 11, 2010-
UpdateApr 17, 2013-
Current statusApr 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j2t
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j2t
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5186_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5186.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the human apoptosome with procaspase-9 CARD
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.26 Å/pix.
x 200 pix.
= 452. Å		2.26 Å/pix.
x 200 pix.
= 452. Å		2.26 Å/pix.
x 200 pix.
= 452. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.26 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-3.31891465 - 5.20101023
Average (Standard dev.)0.03539509 (±0.24957854)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 452.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.262.262.26
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z452.000452.000452.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-3.3195.2010.035

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Supplemental data

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Segmentation: This is a mask used to filter the final 3D volume

AnnotationThis is a mask used to filter the final 3D volume
Fileemd_5186_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human apoptosome with bound procaspase-9 CARD

EntireName: human apoptosome with bound procaspase-9 CARD
Components
  • Sample: human apoptosome with bound procaspase-9 CARD
  • Protein or peptide: Apaf-1
  • Protein or peptide: procaspase-9
  • Protein or peptide: cytochrome c

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Supramolecule #1000: human apoptosome with bound procaspase-9 CARD

SupramoleculeName: human apoptosome with bound procaspase-9 CARD / type: sample / ID: 1000
Details: Apoptosomes were assembled in low salt buffer, procaspase-9 with a thrombin site in the CARD-p20 linker was added, and then the complex was thrombinized to release pc-9 catalytic domains.
Oligomeric state: heptameric Apaf-1 in the apoptosome with 7 bound procaspase-9 CARDs
Number unique components: 3
Molecular weightTheoretical: 1.1 MDa

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Macromolecule #1: Apaf-1

MacromoleculeName: Apaf-1 / type: protein_or_peptide / ID: 1 / Name.synonym: Apaf-1
Details: Seven Apaf-1 molecules were assembled with cytochrome c and dATP to form an apoptosome complex.
Number of copies: 7 / Oligomeric state: heptamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytosol
Molecular weightTheoretical: 135 KDa
Recombinant expressionOrganism: sf21 insect cells (unknown) / Recombinant plasmid: pFastBac1
SequenceGO: GO: 0008635 / InterPro: Apoptotic protease-activating factor 1

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Macromolecule #2: procaspase-9

MacromoleculeName: procaspase-9 / type: protein_or_peptide / ID: 2 / Name.synonym: procaspase-9
Details: procaspase-9 binds on the human apoptosome through CARD-CARD interactions. Procaspase-9 was added to the apoptosome in slight excess and then thrombinized to remove the catalytic domains.
Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytosol
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21b

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Macromolecule #3: cytochrome c

MacromoleculeName: cytochrome c / type: protein_or_peptide / ID: 3 / Name.synonym: cytochrome c / Details: cytochrome c is the assembly activator for Apaf-1 / Number of copies: 7 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: cow / Organelle: mitochrondria
Molecular weightTheoretical: 10 KDa

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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

Concentration3 mg/mL
BufferpH: 7.5
Details: 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT
GridDetails: Quantifoil R1.2/1.3 holey grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III / Details: Vitrification instrument: Vitrobot Mark 3 (FEI) / Method: Blot for 2-3 seconds before plunging at 20C

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 96 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification
DateAug 1, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 400 / Average electron dose: 25 e/Å2 / Od range: 1
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 62000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder.
Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailsparticles were selected with boxer
CTF correctionDetails: Each image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: OTHER / Software - Name: EMAN
Details: Final map contains 34000 particles. Setsf filtration was applied to the final map to boost amplitude at high resolution range and in the last cycles of refinement.
Number images used: 42000
Final two d classificationNumber classes: 592

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Atomic model buiding 1

Initial modelPDB ID:

1z6t


Chain - Chain ID: A
SoftwareName: chimera
DetailsPDBEntryID_givenInChain. Protocol: rigid body for each domain. After chimera fitting, Flex-EM was used to improve fitting and minimize collisions. The beta propellers were modeled using the map as a restraint, sequence alignments and the crystal structure of actin interacting protein 1.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

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

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