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- EMDB-1931: Map of the apoptosome-procaspase-9 complex -

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Basic information

Entry
Database: EMDB / ID: 1931
TitleMap of the apoptosome-procaspase-9 complex
Map dataMap of the apoptosome-procaspase-9 complex
SampleHuman apoptosome-procaspase-9 complex:
Apaf-1APAF1 / Procaspase-9 / Cytochrome c
Keywordsapoptosome / Apaf-1 / procaspase-9 activation
Function / homologyApoptotic protease-activating factor 1
Function and homology information
SourceHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / 16.9 Å resolution
AuthorsYuan S / Ludtke SJ / Akey CW
CitationJournal: Structure / Year: 2011
Title: The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3.
Authors: Shujun Yuan / Xinchao Yu / John M Asara / John E Heuser / Steven J Ludtke / Christopher W Akey
Abstract: Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a ...Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.
DateDeposition: Jul 12, 2011 / Header (metadata) release: Mar 15, 2012 / Map release: Mar 15, 2012 / Last update: Mar 15, 2012

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_1931.map.gz (map file in CCP4 format, 27649 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
192 pix
2.9 Å/pix.
= 556.8 Å
192 pix
2.9 Å/pix.
= 556.8 Å
192 pix
2.9 Å/pix.
= 556.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.9 Å
Density
Contour Level:0.8 (by author), 0.8 (movie #1):
Minimum - Maximum-0.67261672 - 2.2649889
Average (Standard dev.)0.01628205 (0.14226763)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions192192192
Origin000
Limit191191191
Spacing192192192
CellA=B=C: 556.80005 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z556.800556.800556.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.6732.2650.016

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Supplemental data

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Sample components

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Entire Human apoptosome-procaspase-9 complex

EntireName: Human apoptosome-procaspase-9 complex
Details: A slight excess of procaspase-9 was added to ensure saturation of binding to sites on the apoptosome.
Number of components: 3
Oligomeric State: 5-7 procaspase-9 molecules bound to the human apoptosome comprised of 7 Apaf-1 subunits
MassTheoretical: 1.2 MDa

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Component #1: protein, Apaf-1

ProteinName: Apaf-1APAF1 / a.k.a: Apaf-1APAF1 / Oligomeric Details: Heptamer
Details: Apaf-1, cytochrome c and procaspase-9 were co-assembled in the presence of dATP to form the apoptosome-procaspase-9 complex
Number of Copies: 7 / Recombinant expression: Yes
MassTheoretical: 130 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Vector: pFastBac1
Source (natural)Location in cell: Cytosol
External referencesInterPro: Apoptotic protease-activating factor 1

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Component #2: protein, Procaspase-9

ProteinName: Procaspase-9 / a.k.a: Procaspase-9 / Oligomeric Details: Monomer / Recombinant expression: Yes
MassTheoretical: 50 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET21
Source (natural)Location in cell: Cytosol

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Component #3: protein, Cytochrome c

ProteinName: Cytochrome c / a.k.a: Cytochrome c / Oligomeric Details: Monomer / Recombinant expression: No / Number of Copies: 7
MassTheoretical: 10 kDa
SourceSpecies: Bos taurus (cattle)
Source (natural)Location in cell: Mitochondria / Organ or tissue: heart

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/ml
Buffer solution: 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT
pH: 7.5
Support filmQuantifoil holey grids
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 77 K / Humidity: 100 % / Method: Blot for 2-3 seconds before plunging at 20C / Details: Vitrification instrument: Vitrobot Mark 3 (FEI)

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Details: 4k x 4k ccd used
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal), 29000 X (calibrated)
Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1800 - 3500 nm
Specimen HolderHolder: Side entry liquid nitrogen-cooled cryo specimen holder
Model: GATAN LIQUID NITROGEN / Temperature: 96 K
CameraDetector: GENERIC TVIPS

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Image acquisition

Image acquisitionNumber of digital images: 447

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 1000 / Number of projections: 20000
Details: Apaf-1 was co-assembled with bovine cytochrome c and pc-9
Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Projection matching / Software: EMAN2 / CTF correction: Each image
Details: 3D volume was calculated without imposing any symmetry (c1)
Resolution: 16.9 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: chimera / Refinement protocol: rigid body / Target criteria: cross correlation / Refinement space: REAL / Details: Protocol: rigid body
Input PDB model: 3IZA
Modeling #2Software: chimera / Refinement protocol: rigid body / Refinement space: REAL
Details: Protocol: rigid body. final fit was done manually taking into account the orientation of the p20-p10 loop.
Input PDB model: 1JXQ
Chain ID: 1JXQ_A

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