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- EMDB-23302: Cryo-EM structure of NLRP3 double-ring cage, 6-fold (12-mer) -

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Basic information

Entry
Database: EMDB / ID: EMD-23302
TitleCryo-EM structure of NLRP3 double-ring cage, 6-fold (12-mer)
Map dataSharpened map
Sample
  • Complex: NLRP3 double-ring cage, 6-fold (12-mer)
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
KeywordsNLRP3 / NEK7 / nucleotid-binding / ATP-binding / inflammasome / immunity / innate immunity / NACHT / LRR / PYD / IMMUNE SYSTEM
Function / homology
Function and homology information


The NLRP3 inflammasome / positive regulation of T-helper 17 cell differentiation / Metalloprotease DUBs / detection of biotic stimulus / molecular sensor activity / phosphatidylinositol phosphate binding / canonical inflammasome complex / positive regulation of T-helper 2 cell differentiation / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production ...The NLRP3 inflammasome / positive regulation of T-helper 17 cell differentiation / Metalloprotease DUBs / detection of biotic stimulus / molecular sensor activity / phosphatidylinositol phosphate binding / canonical inflammasome complex / positive regulation of T-helper 2 cell differentiation / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production / acute inflammatory response / interphase microtubule organizing center / positive regulation of cytokine production involved in immune response / positive regulation of type 2 immune response / NLRP3 inflammasome complex / leukocyte migration involved in inflammatory response / positive regulation of interleukin-13 production / positive regulation of interleukin-5 production / cellular response to peptidoglycan / osmosensory signaling pathway / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / pattern recognition receptor signaling pathway / pyroptotic inflammatory response / positive regulation of interleukin-4 production / microtubule organizing center / signaling adaptor activity / positive regulation of interleukin-1 beta production / : / ADP binding / protein homooligomerization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / positive regulation of inflammatory response / cellular response to lipopolysaccharide / regulation of inflammatory response / DNA-binding transcription factor binding / defense response to virus / sequence-specific DNA binding / response to ethanol / defense response to Gram-positive bacterium / inflammatory response / Golgi membrane / innate immune response / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / mitochondrion / extracellular region / ATP binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
NACHT-associated domain / Fish-specific NACHT associated domain / Fish-specific NACHT associated 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 / NACHT nucleoside triphosphatase ...NACHT-associated domain / Fish-specific NACHT associated domain / Fish-specific NACHT associated 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 / NACHT nucleoside triphosphatase / NACHT domain / DAPIN domain profile. / NACHT-NTPase domain profile. / PAAD/DAPIN/Pyrin domain / PAAD/DAPIN/Pyrin domain / Leucine rich repeat, ribonuclease inhibitor type / 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 3
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsAndreeva L / Rawson S
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)HD087988 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI124491 United States
Damon Runyon Cancer Research Foundation2376-19 United States
CitationJournal: Cell / Year: 2021
Title: NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation.
Authors: Liudmila Andreeva / Liron David / Shaun Rawson / Chen Shen / Teerithveen Pasricha / Pablo Pelegrin / Hao Wu /
Abstract: The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important ...The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.
History
DepositionJan 17, 2021-
Header (metadata) releaseDec 15, 2021-
Map releaseDec 15, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lfh
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7lfh
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23302.png

Downloads & links

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Map

FileDownload / File: emd_23302.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 422.4 Å		0.83 Å/pix.
x 512 pix.
= 422.4 Å		0.83 Å/pix.
x 512 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum-0.0016653824 - 1.7302464
Average (Standard dev.)0.0022373248 (±0.031246971)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 422.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8250.8250.825
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0021.7300.002

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Supplemental data

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Sample components

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Entire : NLRP3 double-ring cage, 6-fold (12-mer)

EntireName: NLRP3 double-ring cage, 6-fold (12-mer)
Components
  • Complex: NLRP3 double-ring cage, 6-fold (12-mer)
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3

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Supramolecule #1: NLRP3 double-ring cage, 6-fold (12-mer)

SupramoleculeName: NLRP3 double-ring cage, 6-fold (12-mer) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: NLRP3 oligomer
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 1.42 MDa

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Macromolecule #1: NACHT, LRR and PYD domains-containing protein 3

MacromoleculeName: NACHT, LRR and PYD domains-containing protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 118.802219 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GRSAMTSVRC KLAQYLEDLE DVDLKKFKMH LEDYPPEKGC IPVPRGQMEK ADHLDLATLM IDFNGEEKAW AMAVWIFAAI NRRDLWEKA KKDQPEWNDT CTSHSSMVCQ EDSLEEEWMG LLGYLSRISI CKKKKDYCKM YRRHVRSRFY SIKDRNARLG E SVDLNSRY ...String:
GRSAMTSVRC KLAQYLEDLE DVDLKKFKMH LEDYPPEKGC IPVPRGQMEK ADHLDLATLM IDFNGEEKAW AMAVWIFAAI NRRDLWEKA KKDQPEWNDT CTSHSSMVCQ EDSLEEEWMG LLGYLSRISI CKKKKDYCKM YRRHVRSRFY SIKDRNARLG E SVDLNSRY TQLQLVKEHP SKQEREHELL TIGRTKMRDS PMSSLKLELL FEPEDGHSEP VHTVVFQGAA GIGKTILARK IM LDWALGK LFKDKFDYLF FIHCREVSLR TPRSLADLIV SCWPDPNPPV CKILRKPSRI LFLMDGFDEL QGAFDEHIGE VCT DWQKAV RGDILLSSLI RKKLLPKASL LITTRPVALE KLQHLLDHPR HVEILGFSEA KRKEYFFKYF SNELQAREAF RLIQ ENEVL FTMCFIPLVC WIVCTGLKQQ METGKSLAQT SKTTTAVYVF FLSSLLQSRG GIEEHLFSDY LQGLCSLAAD GIWNQ KILF EECDLRKHGL QKTDVSAFLR MNVFQKEVDC ERFYSFSHMT FQEFFAAMYY LLEEEAEGET VRKGPGGCSD LLNRDV KVL LENYGKFEKG YLIFVVRFLF GLVNQERTSY LEKKLSCKIS QQVRLELLKW IEVKAKAKKL QWQPSQLELF YCLYEMQ EE DFVQSAMDHF PKIEINLSTR MDHVVSSFCI KNCHRVKTLS LGFFHNSPKE EEEERRGGRP LDQVQCVFPD THVACSSR L VNCCLTSSFC RGLFSSLSTN RSLTELDLSD NTLGDPGMRV LCEALQHPGC NIQRLWLGRC GLSHQCCFDI SSVLSSSQK LVELDLSDNA LGDFGIRLLC VGLKHLLCNL QKLWLVSCCL TSACCQDLAL VLSSNHSLTR LYIGENALGD SGVQVLCEKM KDPQCNLQK LGLVNSGLTS ICCSALTSVL KTNQNFTHLY LRSNALGDTG LRLLCEGLLH PDCKLQMLEL DNCSLTSHSC W NLSTILTH NHSLRKLNLG NNDLGDLCVV TLCEVLKQQG CLLQSLQLGE MYLNRETKRA LEALQEEKPE LTIVFEISW

UniProtKB: NACHT, LRR and PYD domains-containing protein 3

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.5
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 6800 / Average exposure time: 1.51 sec. / Average electron dose: 53.225 e/Å2
Details: Images were collected as movies with 50 frames, each recorded at multiple defocus values from -0.8 to -2.4 um and with multiple exposures per stage shift (5x4) introduced with image shift.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.4 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6npy


Details: 6NPY was mutated to correspond to mouse sequence. Fragment aa 534-736 was generated from the AlphaFold prediction of mouse NLRP3 structure
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 122941
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6npy


Chain - Chain ID: A / Chain - Residue range: 131-1036 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7lfh:
Cryo-EM structure of NLRP3 double-ring cage, 6-fold (12-mer)

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