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- EMDB-22219: Cryo-EM structure of CARD8-CARD filament -

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Basic information

Entry
Database: EMDB / ID: EMD-22219
TitleCryo-EM structure of CARD8-CARD filament
Map data
Sample
  • Complex: CARD8-CARD filament
    • Protein or peptide: Caspase recruitment domain-containing protein 8
KeywordsFilament / inflammasome / signaling / UPA / FIIND / CARD / NLRP1 / IMMUNE SYSTEM
Function / homology
Function and homology information


CARD8 inflammasome complex assembly / NACHT domain binding / Formation of apoptosome / cysteine-type endopeptidase activator activity / inhibition of cysteine-type endopeptidase activity / negative regulation of NLRP3 inflammasome complex assembly / NLRP3 inflammasome complex / CARD domain binding / negative regulation of lipopolysaccharide-mediated signaling pathway / self proteolysis ...CARD8 inflammasome complex assembly / NACHT domain binding / Formation of apoptosome / cysteine-type endopeptidase activator activity / inhibition of cysteine-type endopeptidase activity / negative regulation of NLRP3 inflammasome complex assembly / NLRP3 inflammasome complex / CARD domain binding / negative regulation of lipopolysaccharide-mediated signaling pathway / self proteolysis / Regulation of the apoptosome activity / Hydrolases; Acting on peptide bonds (peptidases) / regulation of canonical NF-kappaB signal transduction / pattern recognition receptor activity / negative regulation of interleukin-1 beta production / negative regulation of NF-kappaB transcription factor activity / antiviral innate immune response / cysteine-type endopeptidase activator activity involved in apoptotic process / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of canonical NF-kappaB signal transduction / molecular condensate scaffold activity / positive regulation of interleukin-1 beta production / peptidase activity / defense response to virus / regulation of apoptotic process / protein homodimerization activity / protein-containing complex / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
FIIND domain / Function to find / FIIND domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily
Similarity search - Domain/homology
Caspase recruitment domain-containing protein 8
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsHollingsworth LR / David L
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 26, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xkj
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6xkj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22219.png

Downloads & links

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Map

FileDownload / File: emd_22219.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8315 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.047449674 - 0.09074927
Average (Standard dev.)-0.000078693694 (±0.0030161047)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 532.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.83150.83150.8315
M x/y/z640640640
origin x/y/z0.0000.0000.000
length x/y/z532.160532.160532.160
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS640640640
D min/max/mean-0.0470.091-0.000

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Supplemental data

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Sample components

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Entire : CARD8-CARD filament

EntireName: CARD8-CARD filament
Components
  • Complex: CARD8-CARD filament
    • Protein or peptide: Caspase recruitment domain-containing protein 8

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Supramolecule #1: CARD8-CARD filament

SupramoleculeName: CARD8-CARD filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Caspase recruitment domain-containing protein 8

MacromoleculeName: Caspase recruitment domain-containing protein 8 / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.103373 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AAFVKENHRQ LQARMGDLKG VLDDLQDNEV LTENEKELVE QEKTRQSKNE ALLSMVEKKG DLALDVLFRS ISERDPYLVS YLRQQNL

UniProtKB: Caspase recruitment domain-containing protein 8

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3Tris-HClTris
150.0 mMNaClSodium chlorideSodium Chloride
1.0 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.2 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1208 / Average exposure time: 1.25 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 287568 / Software - Name: RELION (ver. 3.1) / Software - details: Manually picked
Startup modelType of model: OTHER
Details: An initial model was built with relion_helix_inimodel2d
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 5.2 Å
Applied symmetry - Helical parameters - Δ&Phi: -99.07 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 111333
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4ikm


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6xkj:
Cryo-EM structure of CARD8-CARD filament

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