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- EMDB-8903: Cryo-EM Structures of ASC and NLRC4 CARD Filaments Reveal a Unifi... -

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Basic information

Entry
Database: EMDB / ID: EMD-8903
TitleCryo-EM Structures of ASC and NLRC4 CARD Filaments Reveal a Unified Mechanism of Nucleation and Activation of Caspase-1
Map dataHelical reconstruction of NLRC4-CARD
Sample
  • Organelle or cellular component: Caspase recruitment domain of NLR family CARD domain-containing protein 4
    • Protein or peptide: NLR family CARD domain-containing protein 4
Function / homology
Function and homology information


IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / caspase binding / positive regulation of protein processing / activation of cysteine-type endopeptidase activity / pattern recognition receptor signaling pathway / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptosis ...IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / caspase binding / positive regulation of protein processing / activation of cysteine-type endopeptidase activity / pattern recognition receptor signaling pathway / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptosis / endopeptidase activator activity / activation of innate immune response / detection of bacterium / positive regulation of interleukin-1 beta production / protein homooligomerization / positive regulation of inflammatory response / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of NF-kappaB transcription factor activity / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / innate immune response / intracellular membrane-bounded organelle / apoptotic process / magnesium ion binding / protein homodimerization activity / ATP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily ...NLR family CARD domain-containing protein 4 / NLRC4, helical domain / NLRC4 helical domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
NLR family CARD domain-containing protein 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsLi Y / Fu T / Wu H
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Cryo-EM structures of ASC and NLRC4 CARD filaments reveal a unified mechanism of nucleation and activation of caspase-1.
Authors: Yang Li / Tian-Min Fu / Alvin Lu / Kristen Witt / Jianbin Ruan / Chen Shen / Hao Wu /
Abstract: Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, ...Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death. Here, we present cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and ASC CARD filaments mediated by conserved three types of asymmetric interactions (types I, II, and III). We find that the CARDs of these two adaptor proteins share a similar assembly pattern, which matches that of the caspase-1 CARD filament whose structure we defined previously. These data indicate a unified mechanism for downstream caspase-1 recruitment through CARD-CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its CARD and its nucleotide-binding domain (NBD), respectively.
History
DepositionJun 12, 2018-
Header (metadata) releaseJun 27, 2018-
Map releaseOct 3, 2018-
UpdateDec 5, 2018-
Current statusDec 5, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6n1i
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6n1i
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8903.png

Downloads & links

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Map

FileDownload / File: emd_8903.map.gz / Format: CCP4 / Size: 45.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of NLRC4-CARD
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 228 pix.
= 300.96 Å		1.32 Å/pix.
x 228 pix.
= 300.96 Å		1.32 Å/pix.
x 228 pix.
= 300.96 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: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.15344454 - 0.26352152
Average (Standard dev.)0.0020723743 (±0.014662194)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-114-114-114
Dimensions228228228
Spacing228228228
CellA=B=C: 300.96002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z228228228
origin x/y/z0.0000.0000.000
length x/y/z300.960300.960300.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS-114-114-114
NC/NR/NS228228228
D min/max/mean-0.1530.2640.002

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Supplemental data

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Sample components

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Entire : Caspase recruitment domain of NLR family CARD domain-containing p...

EntireName: Caspase recruitment domain of NLR family CARD domain-containing protein 4
Components
  • Organelle or cellular component: Caspase recruitment domain of NLR family CARD domain-containing protein 4
    • Protein or peptide: NLR family CARD domain-containing protein 4

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Supramolecule #1: Caspase recruitment domain of NLR family CARD domain-containing p...

SupramoleculeName: Caspase recruitment domain of NLR family CARD domain-containing protein 4
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)

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Macromolecule #1: NLR family CARD domain-containing protein 4

MacromoleculeName: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.987671 KDa
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
SequenceString:
MNFIKDNSRA LIQRMGMTVI KQITDDLFVW NVLNREEVNI ICCEKVEQDA ARGIIHMILK KGSESCNLFL KSLKEWNYPL FQDLN

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 41.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 4.93 Å
Applied symmetry - Helical parameters - Δ&Phi: -100.48 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 199312

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