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- EMDB-11938: Ternary complex of full-length Caspase-8 and FADD -

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Basic information

Entry
Database: EMDB / ID: EMD-11938
TitleTernary complex of full-length Caspase-8 and FADD
Map dataCaspase8:Fadd complex visualised by negative stain EM using single particle analysis
Sample
  • Complex: Ternary complex of full-length Caspase-8 with FADD
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 22.41 Å
AuthorsFox JL / Ragan TJ / Dinsdale D / Fairall L / Schwabe JWR / Morone N / Cain K / MacFarlane M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.
Authors: Joanna L Fox / Michelle A Hughes / Xin Meng / Nikola A Sarnowska / Ian R Powley / Rebekah Jukes-Jones / David Dinsdale / Timothy J Ragan / Louise Fairall / John W R Schwabe / Nobuhiro Morone ...Authors: Joanna L Fox / Michelle A Hughes / Xin Meng / Nikola A Sarnowska / Ian R Powley / Rebekah Jukes-Jones / David Dinsdale / Timothy J Ragan / Louise Fairall / John W R Schwabe / Nobuhiro Morone / Kelvin Cain / Marion MacFarlane /
Abstract: Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD: ...Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIP into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.
History
DepositionNov 16, 2020-
Header (metadata) releaseFeb 17, 2021-
Map releaseFeb 17, 2021-
UpdateFeb 17, 2021-
Current statusFeb 17, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11938.png

Downloads & links

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Map

FileDownload / File: emd_11938.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCaspase8:Fadd complex visualised by negative stain EM using single particle analysis
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.63 Å/pix.
x 128 pix.
= 720. Å		5.63 Å/pix.
x 128 pix.
= 720. Å		5.63 Å/pix.
x 128 pix.
= 720. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.625 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.3 / Movie #1: 2.3
Minimum - Maximum-2.4515731 - 8.461516
Average (Standard dev.)-0.048679136 (±0.5003174)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 720.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.6255.6255.625
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z720.000720.000720.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-2.4528.462-0.049

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Supplemental data

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Sample components

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Entire : Ternary complex of full-length Caspase-8 with FADD

EntireName: Ternary complex of full-length Caspase-8 with FADD
Components
  • Complex: Ternary complex of full-length Caspase-8 with FADD

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Supramolecule #1: Ternary complex of full-length Caspase-8 with FADD

SupramoleculeName: Ternary complex of full-length Caspase-8 with FADD / type: complex / ID: 1 / Parent: 0
Details: Proteins co-expressed and co-purified with FLAG affinity tag
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F / Recombinant plasmid: pCDNA3

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.8
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: Negatively stained EM specimens were prepared following incubation of the sample on the grid overnight at 4 degrees

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Number grids imaged: 1 / Number real images: 620 / Average exposure time: 1.0 sec. / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 57000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3200
Details: Complexes were manually picked from the micrographs
CTF correctionSoftware - Name: Gctf / Software - details: correction applied by RELION
Startup modelType of model: INSILICO MODEL / In silico model: Initial model was generated by CRYOSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 22.41 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1)
Details: Heterogenous refinement was used for the final reconstruction
Number images used: 1002
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 1)

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Atomic model buiding 1

Initial modelPDB ID:

5l08

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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