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

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Basic information

Entry
Database: EMDB / ID: EMD-9137
TitleCryo-EM structure of NLRC4-CARD filament
Map data
Sample
  • Complex: NLRC4-CARD filament
    • Protein or peptide: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
KeywordsNLRC4 / Helical assembly / Inflammasome / PROTEIN FIBRIL
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 / pattern recognition receptor signaling pathway / activation of cysteine-type endopeptidase activity / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptotic inflammatory response ...IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / caspase binding / positive regulation of protein processing / pattern recognition receptor signaling pathway / activation of cysteine-type endopeptidase activity / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptotic inflammatory response / endopeptidase activator activity / detection of bacterium / activation of innate immune response / bioluminescence / positive regulation of interleukin-1 beta production / generation of precursor metabolites and energy / protein homooligomerization / positive regulation of inflammatory response / positive regulation of NF-kappaB transcription factor activity / defense response to bacterium / inflammatory response / positive regulation of apoptotic process / intracellular membrane-bounded organelle / innate immune response / 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 / Nlrc4-like, winged helix domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. ...NLR family CARD domain-containing protein 4 / NLRC4, helical domain / : / NLRC4 helical domain / Nlrc4-like, winged helix domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Death-like domain superfamily / Green fluorescent protein / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
EGFP / NLR family CARD domain-containing protein 4
Similarity search - Component
Biological speciesHomo sapiens (human) / Vaccinia virus
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZheng W / Matyszewski M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM0352569 United States
American Cancer SocietyRG-15-224 United States
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 upstream and downstream CARD assemblies in inflammasomes. Furthermore, a Monte Carlo simulation of the NLRC4 filament assembly rationalized 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.
History
DepositionSep 26, 2018-
Header (metadata) releaseOct 3, 2018-
Map releaseNov 7, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

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

emd_9137.png

Downloads & links

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Map

FileDownload / File: emd_9137.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 128 pix.
= 168.96 Å		1.32 Å/pix.
x 128 pix.
= 168.96 Å		1.32 Å/pix.
x 128 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00032 / Movie #1: 0.00032
Minimum - Maximum-0.00043216487 - 0.0011394405
Average (Standard dev.)0.00007735804 (±0.00010056644)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 168.96 Å
α=β=γ: 90.0 °

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
Components
  • Complex: NLRC4-CARD filament
    • Protein or peptide: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP

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Supramolecule #1: NLRC4-CARD filament

SupramoleculeName: NLRC4-CARD filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Chimera protein of NLR family CARD domain-containing protein 4 an...

MacromoleculeName: Chimera protein of NLR family CARD domain-containing protein 4 and EGFP
type: protein_or_peptide / ID: 1 / Number of copies: 31 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus
Molecular weightTheoretical: 38.668805 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GTGMNFIKDN SRALIQRMGM TVIKQITDDL FVWNVLNREE VNIICCEKVE QDAARGIIHM ILKKGSESCN LFLKSLKEWN YPLFQDLNG QSLFHQTSEG KLMVSKGEEL FTGVVPILVE LDGDVNGHKF SVSGEGEGDA TYGKLTLKFI CTTGKLPVPW P TLVTTLTY ...String:
GTGMNFIKDN SRALIQRMGM TVIKQITDDL FVWNVLNREE VNIICCEKVE QDAARGIIHM ILKKGSESCN LFLKSLKEWN YPLFQDLNG QSLFHQTSEG KLMVSKGEEL FTGVVPILVE LDGDVNGHKF SVSGEGEGDA TYGKLTLKFI CTTGKLPVPW P TLVTTLTY GVQCFSRYPD HMKQHDFFKS AMPEGYVQER TIFFKDDGNY KTRAEVKFEG DTLVNRIELK GIDFKEDGNI LG HKLEYNY NSHNVYIMAD KQKNGIKANF KIRHNIEDGS VQLADHYQQN TPIGDGPVLL PDNHYLSTQS ALSKDPNEKR DHM VLLEFV TAAGITLEHH HHHH

UniProtKB: NLR family CARD domain-containing protein 4, EGFP

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
Details: 20mM HEPES at pH 7.4, 400mM NaCl, 10% glycerol, 1mM EDTA and 1mM DTT
GridMaterial: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1690 / Average exposure time: 12.0 sec. / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 5.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 100.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPIDER / Number images used: 299537
Segment selectionNumber selected: 402078 / Software - Name: EMAN2
Startup modelType of model: OTHER / Details: Featureless cylinder
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial modelPDB ID:

4ikm


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

PDB-6mks:
Cryo-EM structure of NLRC4-CARD filament

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