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- EMDB-9137: Cryo-EM structure of NLRC4-CARD filament -

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Entry
Database: EMDB / ID: 9137
TitleCryo-EM structure of NLRC4-CARD filament
Map data
SampleNLRC4-CARD filament
  • Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
Function / homologyCaspase recruitment domain / Green fluorescent protein / Green fluorescent protein / NACHT domain / CARD caspase recruitment domain profile. / Green fluorescent protein, GFP / CARD domain / NACHT nucleoside triphosphatase / Death-like domain superfamily / Green fluorescent protein-related ...Caspase recruitment domain / Green fluorescent protein / Green fluorescent protein / NACHT domain / CARD caspase recruitment domain profile. / Green fluorescent protein, GFP / CARD domain / NACHT nucleoside triphosphatase / Death-like domain superfamily / Green fluorescent protein-related / NACHT-NTPase domain profile. / TP53 Regulates Transcription of Caspase Activators and Caspases / P-loop containing nucleoside triphosphate hydrolase / The IPAF inflammasome / Leucine-rich repeat domain superfamily / interleukin-1 beta secretion / IPAF inflammasome complex / pyroptosis / inhibition of cysteine-type endopeptidase activity involved in apoptotic process / detection of bacterium / activation of innate immune response / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / regulation of signal transduction / bioluminescence / generation of precursor metabolites and energy / activation of cysteine-type endopeptidase activity involved in apoptotic process / ubiquitin-protein transferase activity / protein homooligomerization / regulation of apoptotic process / positive regulation of NF-kappaB transcription factor activity / inflammatory response / positive regulation of apoptotic process / intracellular / defense response to bacterium / innate immune response / magnesium ion binding / protein homodimerization activity / ATP binding / identical protein binding / nucleus / cytosol / EGFP / NLR family CARD domain-containing protein 4
Function and homology information
SourceHomo sapiens (human) / Vaccinia virus
Methodhelical reconstruction / cryo EM / 3.4 Å resolution
AuthorsZheng W / Matyszewski M / Sohn J / Egelman EH
CitationJournal: J. Biol. Chem. / Year: 2018
Title: Cryo-EM structure of the NLRC4 filament provides insights into how symmetric and asymmetric supramolecular structures drive inflammasome assembly.
Authors: Mariusz Matyszewski / Weili Zheng / Jacob Lueck / Brendan Antiochos / Edward H Egelman / Jungsan Sohn
Abstract: Inflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The Nod Like Receptor (NLR) family apoptosis inhibitory protein (NAIP)•NLR family ...Inflammasomes are supramolecular signaling platforms integral to innate immune defense against invading pathogens. The Nod Like Receptor (NLR) family apoptosis inhibitory protein (NAIP)•NLR family caspase-recruiting domain (CARD) domain-containing 4 (NLRC4) inflammasome recognizes intracellular bacteria and induces the polymerization of the caspase-1 protease, which in turn executes maturation of interleukin-1β (IL-1β) and pyroptosis. Several high-resolution structures of the fully assembled NAIP•NLRC4 complex are available, but these structures do not resolve the architecture of the CARD filament in atomic detail. Here, we present the cryo-EM structure of the filament assembled by the CARD of human NLRC4 (NLRC4) at 3.4 Å resolution. The structure revealed that the helical architecture of the NLRC4 filament is essentially identical to that of the downstream filament assembled by the CARD of caspase-1 (casp1), but deviates from the split washer-like assembly of the NAIP•NLRC4 oligomer. Our results suggest that architectural complementarity is a major driver for the recognition between up- and downstream CARD assemblies in inflammasomes. Furthermore, a Monte Carlo simulation of the NLRC4 filament assembly rationalizes why an (un)decameric NLRC4 oligomer is optimal for assembling the helical base of the NLRC4 filament. Together, our results explain how symmetric and asymmetric supramolecular assemblies enable high-fidelity signaling in inflammasomes.
Validation ReportPDB-ID: 6mks

SummaryFull reportAbout validation report
DateDeposition: Sep 26, 2018 / Header (metadata) release: Oct 3, 2018 / Map release: Nov 7, 2018 / Last update: Nov 14, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00032
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00032
  • Imaged by UCSF Chimera
  • Download
  • Map surface with fitted models
  • Surface level: 0.00032
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6mks
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9137.map.gz (map file in CCP4 format, 8389 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
1.32 Å/pix.
= 168.96 Å
128 pix
1.32 Å/pix.
= 168.96 Å
128 pix
1.32 Å/pix.
= 168.96 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
Contour Level:0.00032 (by author), 0.00032 (movie #1):
Minimum - Maximum-0.00043216487 - 0.0011394405
Average (Standard dev.)0.00007735804 (0.00010056644)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin-64.0-64.0-64.0
Limit63.063.063.0
Spacing128128128
CellA=B=C: 168.96 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z168.960168.960168.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0000.0010.000

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Supplemental data

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Sample components

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Entire NLRC4-CARD filament

EntireName: NLRC4-CARD filament / Number of components: 2

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Component #1: protein, NLRC4-CARD filament

ProteinName: NLRC4-CARD filament / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pET21b

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Component #2: protein, Chimera protein of NLR family CARD domain-containing pro...

ProteinName: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
Number of Copies: 31 / Recombinant expression: No
MassTheoretical: 38.668805 kDa
SourceSpecies: Vaccinia virus
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 5 Å / Delta phi: 100.6 deg.
Sample solutionBuffer solution: 20mM HEPES at pH 7.4, 400mM NaCl, 10% glycerol, 1mM EDTA and 1mM DTT
pH: 7
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

ImagingMicroscope: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 42 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1690

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: BACK PROJECTION / Software: SPIDER / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Input PDB model: 4IKM
Output model

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