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- EMDB-6480: Atomic structure of the Apaf-1 apoptosome -

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Basic information

Entry
Database: EMDB / ID: EMD-6480
TitleAtomic structure of the Apaf-1 apoptosome
Map dataReconstruction of the Apaf-1 apoptosome
Sample
  • Sample: Heptameric apoptosome of activated human Apaf-1 binding to horse cytochrome c and dATP
  • Protein or peptide: The apoptotic protease activating factor 1
  • Protein or peptide: cytochrome c
KeywordsApoptosome / cryo-EM structure / apoptosis / Apaf-1
Function / homology
Function and homology information


response to G1 DNA damage checkpoint signaling / : / Formation of apoptosome / regulation of apoptotic DNA fragmentation / apoptosome / cytochrome complex / : / cytochrome c-heme linkage / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity ...response to G1 DNA damage checkpoint signaling / : / Formation of apoptosome / regulation of apoptotic DNA fragmentation / apoptosome / cytochrome complex / : / cytochrome c-heme linkage / 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 / forebrain development / cellular response to transforming growth factor beta stimulus / cardiac muscle cell apoptotic process / heat shock protein binding / intrinsic apoptotic signaling pathway / response to nutrient / neural tube closure / kidney development / positive regulation of apoptotic signaling pathway / : / mitochondrial intermembrane space / ADP binding / nervous system development / secretory granule lumen / regulation of apoptotic process / neuron apoptotic process / ficolin-1-rich granule lumen / cell differentiation / electron transfer activity / response to hypoxia / positive regulation of apoptotic process / nucleotide binding / lipid 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 / 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. / 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) / Equus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhou MY / Li YN / Hu Q / Bai XC / Huang WY / Yan CY / Scheres SHW / Shi YG
CitationJournal: Genes Dev / Year: 2015
Title: Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.
Authors: Mengying Zhou / Yini Li / Qi Hu / Xiao-Chen Bai / Weiyun Huang / Chuangye Yan / Sjors H W Scheres / Yigong Shi /
Abstract: The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding ...The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.
History
DepositionOct 14, 2015-
Header (metadata) releaseNov 18, 2015-
Map releaseNov 18, 2015-
UpdateNov 18, 2015-
Current statusNov 18, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbt
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbt
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6480.gif

Downloads & links

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Map

FileDownload / File: emd_6480.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the Apaf-1 apoptosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 320 pix.
= 422.4 Å		1.32 Å/pix.
x 320 pix.
= 422.4 Å		1.32 Å/pix.
x 320 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.23910286 - 0.35140538
Average (Standard dev.)0.00003032 (±0.00817638)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2390.3510.000

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Supplemental data

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Sample components

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Entire : Heptameric apoptosome of activated human Apaf-1 binding to horse ...

EntireName: Heptameric apoptosome of activated human Apaf-1 binding to horse cytochrome c and dATP
Components
  • Sample: Heptameric apoptosome of activated human Apaf-1 binding to horse cytochrome c and dATP
  • Protein or peptide: The apoptotic protease activating factor 1
  • Protein or peptide: cytochrome c

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Supramolecule #1000: Heptameric apoptosome of activated human Apaf-1 binding to horse ...

SupramoleculeName: Heptameric apoptosome of activated human Apaf-1 binding to horse cytochrome c and dATP
type: sample / ID: 1000 / Oligomeric state: Heptamer / Number unique components: 2

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Macromolecule #1: The apoptotic protease activating factor 1

MacromoleculeName: The apoptotic protease activating factor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: Apaf-1 / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High Five / Recombinant plasmid: pFastBac
SequenceUniProtKB: 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 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Equus caballus (horse) / synonym: Horse
SequenceUniProtKB: Cytochrome 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

BufferpH: 7.5
Details: 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA and 1 mM DTT
GridDetails: 400-mesh Cu R 1.2/1.3 grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateJan 10, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 912 / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.4 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 134919

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Atomic model buiding 1

Initial modelPDB ID:

3j2t

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jbt:
Atomic structure of the Apaf-1 apoptosome

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