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- EMDB-9946: Structure of NLRC4 CARD filament -

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Basic information

Entry
Database: EMDB / ID: EMD-9946
TitleStructure of NLRC4 CARD filament
Map dataNone
Sample
  • Complex: NLRC4 CARD filament
    • Protein or peptide: NLR family CARD domain-containing protein 4
Keywordsfilament / SIGNALING PROTEIN
Function / homology
Function and homology information


IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / positive regulation of protein processing / caspase binding / activation of cysteine-type endopeptidase activity / pattern recognition receptor signaling pathway / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptotic inflammatory response ...IPAF inflammasome complex / The IPAF inflammasome / icosanoid biosynthetic process / canonical inflammasome complex / positive regulation of protein processing / caspase binding / activation of cysteine-type endopeptidase activity / pattern recognition receptor signaling pathway / TP53 Regulates Transcription of Caspase Activators and Caspases / pyroptotic inflammatory response / endopeptidase activator activity / detection of bacterium / activation of innate immune response / 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 / 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 / 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: 3.3 Å
AuthorsGong Q / Xu C
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (Singapore)tier2 and tier1 Singapore
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8.
Authors: Qin Gong / Kim Robinson / Chenrui Xu / Phuong Thao Huynh / Kelvin Han Chung Chong / Eddie Yong Jun Tan / Jiawen Zhang / Zhao Zhi Boo / Daniel Eng Thiam Teo / Kenneth Lay / Yaming Zhang / ...Authors: Qin Gong / Kim Robinson / Chenrui Xu / Phuong Thao Huynh / Kelvin Han Chung Chong / Eddie Yong Jun Tan / Jiawen Zhang / Zhao Zhi Boo / Daniel Eng Thiam Teo / Kenneth Lay / Yaming Zhang / John Soon Yew Lim / Wah Ing Goh / Graham Wright / Franklin L Zhong / Bruno Reversade / Bin Wu /
Abstract: Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 ...Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIIND-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIIND-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIIND. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIIND which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.
History
DepositionJun 12, 2019-
Header (metadata) releaseSep 16, 2020-
Map releaseSep 16, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0164
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0164
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6k8j
  • Surface level: 0.0164
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6k8j
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9946.png

Downloads & links

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Map

FileDownload / File: emd_9946.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0164 / Movie #1: 0.0164
Minimum - Maximum-0.006646438 - 0.03191511
Average (Standard dev.)0.0038547025 (±0.0056631668)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin585853
Dimensions888989
Spacing898889
CellA: 97.9 Å / B: 96.8 Å / C: 97.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z898889
origin x/y/z0.0000.0000.000
length x/y/z97.90096.80097.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS585853
NC/NR/NS898889
D min/max/mean-0.0070.0320.004

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Supplemental data

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Additional map: cropped 12mer from filament, used for model alignment...

Fileemd_9946_additional.map
Annotationcropped 12mer from filament, used for model alignment and refinement, not for publication
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_9946_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_9946_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: NLR family CARD domain-containing protein 4

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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: NLR family CARD domain-containing protein 4

MacromoleculeName: NLR family CARD domain-containing protein 4 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.600412 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
GPGVDEALRE AQTKSAMNFI KDNSRALIQR MGMTVIKQIT DDLFVWNVLN REEVNIICCE KVEQDAARGI IHMILKKGSE SCNLFLKSL KEWNYPLFQD LN

UniProtKB: NLR family CARD domain-containing protein 4

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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: 8
GridMaterial: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 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: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 2109 / 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: 382715 / Software - Name: RELION (ver. 2.0)
Startup modelType of model: OTHER / Details: solid column
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Helical parameters - Δz: 5.17 Å
Applied symmetry - Helical parameters - Δ&Phi: -100.55 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 120602
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6k8j:
Structure of NLRC4 CARD filament

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