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- EMDB-22220: Cryo-EM structure of the NLRP1-CARD filament -

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Basic information

Entry
Database: EMDB / ID: EMD-22220
TitleCryo-EM structure of the NLRP1-CARD filament
Map dataCryo-EM of NLRP1-CARD filament
Sample
  • Complex: NLRP1-CARD filament
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 1
KeywordsFilament / inflammasome / signaling / UPA / CARD / FIIND / NLRP1 / IMMUNE SYSTEM
Function / homology
Function and homology information


NLRP1 inflammasome complex assembly / NLRP1 inflammasome complex / cysteine-type endopeptidase activator activity / The NLRP1 inflammasome / NLRP3 inflammasome complex / self proteolysis / Hydrolases; Acting on peptide bonds (peptidases) / pattern recognition receptor signaling pathway / cellular response to UV-B / pattern recognition receptor activity ...NLRP1 inflammasome complex assembly / NLRP1 inflammasome complex / cysteine-type endopeptidase activator activity / The NLRP1 inflammasome / NLRP3 inflammasome complex / self proteolysis / Hydrolases; Acting on peptide bonds (peptidases) / pattern recognition receptor signaling pathway / cellular response to UV-B / pattern recognition receptor activity / pyroptotic inflammatory response / cysteine-type endopeptidase activator activity involved in apoptotic process / response to muramyl dipeptide / signaling adaptor activity / : / antiviral innate immune response / activation of innate immune response / intrinsic apoptotic signaling pathway / positive regulation of interleukin-1 beta production / molecular condensate scaffold activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein homooligomerization / positive regulation of inflammatory response / double-stranded RNA binding / peptidase activity / double-stranded DNA binding / regulation of inflammatory response / regulation of apoptotic process / neuron apoptotic process / defense response to virus / defense response to bacterium / inflammatory response / protein domain specific binding / apoptotic process / nucleolus / enzyme binding / signal transduction / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
FIIND domain / Function to find / FIIND domain profile. / CARD8/ASC/NALP1, CARD domain / : / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / DAPIN domain ...FIIND domain / Function to find / FIIND domain profile. / CARD8/ASC/NALP1, CARD domain / : / NACHT, LRR and PYD domains-containing protein, helical domain HD2 / NLRC4 helical domain HD2 / NOD2, winged helix domain / NOD2 winged helix domain / DAPIN domain / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / Leucine rich repeat, ribonuclease inhibitor type / Leucine Rich repeat / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Leucine Rich Repeat / Death-like domain superfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
NACHT, LRR and PYD domains-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.72 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-6xkk
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6xkk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22220.png

Downloads & links

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Map

FileDownload / File: emd_22220.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM of NLRP1-CARD filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.51 Å/pix.
x 360 pix.
= 542.88 Å		1.51 Å/pix.
x 360 pix.
= 542.88 Å		1.51 Å/pix.
x 360 pix.
= 542.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.508 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.037116013 - 0.0868414
Average (Standard dev.)0.000013651564 (±0.0037921434)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 542.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5081.5081.508
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z542.880542.880542.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0370.0870.000

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Supplemental data

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Sample components

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Entire : NLRP1-CARD filament

EntireName: NLRP1-CARD filament
Components
  • Complex: NLRP1-CARD filament
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 1

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Supramolecule #1: NLRP1-CARD filament

SupramoleculeName: NLRP1-CARD filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: NACHT, LRR and PYD domains-containing protein 1

MacromoleculeName: NACHT, LRR and PYD domains-containing protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 44 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.138815 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
LHFVDQYREQ LIARVTSVEV VLDKLHGQVL SQEQYERVLA ENTRPSQMRK LFSLSQSWDR KCKDGLYQAL KETHPHLIME LWEKGSKKG LLPLSS

UniProtKB: NACHT, LRR and PYD domains-containing protein 1

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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-HCl
150.0 mMNaClSodium Chloride
1.0 mMC9H15O6PTCEP
GridModel: C-flat-1.2/1.3 / 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 IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 25 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 1991 / Average exposure time: 3.5 sec. / Average electron dose: 52.3 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 / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 5.1 Å
Applied symmetry - Helical parameters - Δ&Phi: -100.8 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 18272
Segment selectionNumber selected: 118006
Startup modelType of model: OTHER
Details: An initial model was built with relion_helix_inimodel2d
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4ifp


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

PDB-6xkk:
Cryo-EM structure of the NLRP1-CARD filament

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