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- EMDB-22233: Cryo-EM structure of ASC-Caspase1 Octamer -

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Basic information

Entry
Database: EMDB / ID: EMD-22233
TitleCryo-EM structure of ASC-Caspase1 Octamer
Map data
Sample
  • Complex: Cryo-EM structure of ASC-Caspase1 Octamer
Function / homology
Function and homology information


NLRP6 inflammasome complex / myosin I binding / Pyrin domain binding / myeloid dendritic cell activation involved in immune response / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / negative regulation of protein serine/threonine kinase activity / regulation of intrinsic apoptotic signaling pathway / caspase-1 / protease inhibitor complex / myeloid dendritic cell activation ...NLRP6 inflammasome complex / myosin I binding / Pyrin domain binding / myeloid dendritic cell activation involved in immune response / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / negative regulation of protein serine/threonine kinase activity / regulation of intrinsic apoptotic signaling pathway / caspase-1 / protease inhibitor complex / myeloid dendritic cell activation / AIM2 inflammasome complex assembly / IkappaB kinase complex / The AIM2 inflammasome / AIM2 inflammasome complex / IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / NLRP1 inflammasome complex / macropinocytosis / canonical inflammasome complex / interleukin-6 receptor binding / positive regulation of interleukin-18 production / NLRP3 inflammasome complex assembly / BMP receptor binding / positive regulation of adaptive immune response / CARD domain binding / cytokine precursor processing / NLRP3 inflammasome complex / cysteine-type endopeptidase activator activity / CLEC7A/inflammasome pathway / osmosensory signaling pathway / negative regulation of interferon-beta production / Interleukin-1 processing / regulation of tumor necrosis factor-mediated signaling pathway / Interleukin-37 signaling / positive regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of extrinsic apoptotic signaling pathway / pattern recognition receptor signaling pathway / cysteine-type endopeptidase activator activity involved in apoptotic process / positive regulation of macrophage cytokine production / signaling receptor ligand precursor processing / negative regulation of NF-kappaB transcription factor activity / pattern recognition receptor activity / TP53 Regulates Transcription of Caspase Activators and Caspases / tropomyosin binding / pyroptotic inflammatory response / cytokine binding / positive regulation of actin filament polymerization / positive regulation of release of cytochrome c from mitochondria / positive regulation of activated T cell proliferation / protein autoprocessing / intrinsic apoptotic signaling pathway by p53 class mediator / The NLRP3 inflammasome / positive regulation of interleukin-10 production / cellular response to interleukin-1 / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / Pyroptosis / positive regulation of T cell migration / positive regulation of DNA-binding transcription factor activity / Purinergic signaling in leishmaniasis infection / positive regulation of chemokine production / positive regulation of defense response to virus by host / negative regulation of canonical NF-kappaB signal transduction / negative regulation of cytokine production involved in inflammatory response / activation of innate immune response / intrinsic apoptotic signaling pathway / positive regulation of phagocytosis / protein maturation / positive regulation of interleukin-1 beta production / tumor necrosis factor-mediated signaling pathway / positive regulation of interleukin-8 production / positive regulation of JNK cascade / apoptotic signaling pathway / cellular response to mechanical stimulus / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of NF-kappaB transcription factor activity / NOD1/2 Signaling Pathway / protein homooligomerization / positive regulation of T cell activation / regulation of protein stability / cellular response to type II interferon / positive regulation of interleukin-6 production / kinase binding / positive regulation of type II interferon production / positive regulation of inflammatory response / positive regulation of tumor necrosis factor production / SARS-CoV-1 activates/modulates innate immune responses / cellular response to tumor necrosis factor / azurophil granule lumen / cellular response to lipopolysaccharide / regulation of inflammatory response / protease binding / secretory granule lumen / regulation of apoptotic process / endopeptidase activity / defense response to Gram-negative bacterium / defense response to virus / microtubule / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade
Similarity search - Function
CARD8/ASC/NALP1, CARD domain / : / Caspase recruitment domain / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain ...CARD8/ASC/NALP1, CARD domain / : / Caspase recruitment domain / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / 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 / Caspase-like domain superfamily / Death-like domain superfamily
Similarity search - Domain/homology
Caspase-1 / Apoptosis-associated speck-like protein containing a CARD
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsHollingsworth LR / David L / Li Y / Ruan J / Wu H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director1DP1 HD087988 United States
CitationJournal: Nat Commun / Year: 2021
Title: Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.
Authors: L Robert Hollingsworth / Liron David / Yang Li / Andrew R Griswold / Jianbin Ruan / Humayun Sharif / Pietro Fontana / Elizabeth L Orth-He / Tian-Min Fu / Daniel A Bachovchin / Hao Wu /
Abstract: NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation ...NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1 filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASC-caspase-1 octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.
History
DepositionJun 27, 2020-
Header (metadata) releaseDec 9, 2020-
Map releaseDec 9, 2020-
UpdateDec 22, 2021-
Current statusDec 22, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0439
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0439
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7keu
  • Surface level: 0.0439
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22233.png

Downloads & links

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Map

FileDownload / File: emd_22233.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.65 Å/pix.
x 120 pix.
= 198. Å		1.65 Å/pix.
x 120 pix.
= 198. Å		1.65 Å/pix.
x 120 pix.
= 198. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0439 / Movie #1: 0.0439
Minimum - Maximum-0.0800307 - 0.19025669
Average (Standard dev.)0.00057472993 (±0.006224771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 198.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.651.651.65
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z198.000198.000198.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.0800.1900.001

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of ASC-Caspase1 Octamer

EntireName: Cryo-EM structure of ASC-Caspase1 Octamer
Components
  • Complex: Cryo-EM structure of ASC-Caspase1 Octamer

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Supramolecule #1: Cryo-EM structure of ASC-Caspase1 Octamer

SupramoleculeName: Cryo-EM structure of ASC-Caspase1 Octamer / type: complex / ID: 1 / Parent: 0
Details: A complex of ASC-CARD tetramer and Caspase1-CARD tetramer
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 86.3 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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3Tris-HCl
150.0 mMNaClSodium Chloride
2.0 mMDTT
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1606 / Average electron dose: 58.0 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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 351257 / Details: Particles were picked with samautopick.py
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: An initial model of 4 ASC-CARD and 4 Caspase1-CARD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 89861
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

5fna

,

3j63

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RefinementProtocol: RIGID BODY FIT
Output model

PDB-7keu:
Cryo-EM structure of the Caspase-1-CARD:ASC-CARD octamer

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