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- EMDB-23303: Cryo-EM structure of NLRP3 double-ring cage, 6-fold (12-mer) -

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Basic information

Entry
Database: EMDB / ID: EMD-23303
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
Function / homology
Function and homology information


The NLRP3 inflammasome / positive regulation of T-helper 17 cell differentiation / small molecule sensor activity / detection of biotic stimulus / Metalloprotease DUBs / phosphatidylinositol phosphate binding / positive regulation of cytokine production involved in immune response / positive regulation of T-helper 2 cell differentiation / canonical inflammasome complex / NLRP3 inflammasome complex assembly ...The NLRP3 inflammasome / positive regulation of T-helper 17 cell differentiation / small molecule sensor activity / detection of biotic stimulus / Metalloprotease DUBs / phosphatidylinositol phosphate binding / positive regulation of cytokine production involved in immune response / positive regulation of T-helper 2 cell differentiation / canonical inflammasome complex / NLRP3 inflammasome complex assembly / interphase microtubule organizing center / acute inflammatory response / positive regulation of T-helper 2 cell cytokine production / 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 / microtubule organizing center / negative regulation of NF-kappaB transcription factor activity / positive regulation of interleukin-4 production / pyroptotic inflammatory response / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / negative regulation of acute inflammatory response / signaling adaptor activity / positive regulation of interleukin-1 beta production / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / protein homooligomerization / response to organic cyclic compound / ADP binding / positive regulation of inflammatory response / regulation of inflammatory response / cellular response to lipopolysaccharide / DNA-binding transcription factor binding / response to ethanol / defense response to virus / sequence-specific DNA binding / defense response to Gram-positive bacterium / inflammatory response / Golgi membrane / innate immune response / endoplasmic reticulum / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular region / ATP binding / membrane / nucleus / cytoplasm / cytosol
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 / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain ...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 / DAPIN domain profile. / PAAD/DAPIN/Pyrin domain / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / PAAD/DAPIN/Pyrin domain / Leucine rich repeat, ribonuclease inhibitor type / Leucine Rich repeat / Leucine-rich repeat profile. / Death-like domain superfamily / 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: 5.8 Å
AuthorsAndreeva L / Rawson S / Wu H
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-
UpdateJan 5, 2022-
Current statusJan 5, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.16
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.16
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23303.png

Downloads & links

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Map

FileDownload / File: emd_23303.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 1.65 Å
Density
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.16
Minimum - Maximum-0.0017368507 - 1.8825066
Average (Standard dev.)0.0013013949 (±0.023782792)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 528.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.651.651.65
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z528.000528.000528.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0021.8830.001

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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)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T
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 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GRSAMTSVRC KLAQYLEDLE DVDLKKFKMH LEDYPPEKGC IPVPRGQMEK ADHLDLATLM IDFNGEEKAW AMAVWIFAAI NRRDLWEKAK KDQPEWNDTC TSHSSMVCQE DSLEEEWMGL LGYLSRISIC KKKKDYCKMY RRHVRSRFYS IKDRNARLGE SVDLNSRYTQ ...String:
GRSAMTSVRC KLAQYLEDLE DVDLKKFKMH LEDYPPEKGC IPVPRGQMEK ADHLDLATLM IDFNGEEKAW AMAVWIFAAI NRRDLWEKAK KDQPEWNDTC TSHSSMVCQE DSLEEEWMGL LGYLSRISIC KKKKDYCKMY RRHVRSRFYS IKDRNARLGE SVDLNSRYTQ LQLVKEHPSK QEREHELLTI GRTKMRDSPM SSLKLELLFE PEDGHSEPVH TVVFQGAAGI GKTILARKIM LDWALGKLFK DKFDYLFFIH CREVSLRTPR SLADLIVSCW PDPNPPVCKI LRKPSRILFL MDGFDELQGA FDEHIGEVCT DWQKAVRGDI LLSSLIRKKL LPKASLLITT RPVALEKLQH LLDHPRHVEI LGFSEAKRKE YFFKYFSNEL QAREAFRLIQ ENEVLFTMCF IPLVCWIVCT GLKQQMETGK SLAQTSKTTT AVYVFFLSSL LQSRGGIEEH LFSDYLQGLC SLAADGIWNQ KILFEECDLR KHGLQKTDVS AFLRMNVFQK EVDCERFYSF SHMTFQEFFA AMYYLLEEEA EGETVRKGPG GCSDLLNRDV KVLLENYGKF EKGYLIFVVR FLFGLVNQER TSYLEKKLSC KISQQVRLEL LKWIEVKAKA KKLQWQPSQL ELFYCLYEMQ EEDFVQSAMD HFPKIEINLS TRMDHVVSSF CIKNCHRVKT LSLGFFHNSP KEEEEERRGG RPLDQVQCVF PDTHVACSSR LVNCCLTSSF CRGLFSSLST NRSLTELDLS DNTLGDPGMR VLCEALQHPG CNIQRLWLGR CGLSHQCCFD ISSVLSSSQK LVELDLSDNA LGDFGIRLLC VGLKHLLCNL QKLWLVSCCL TSACCQDLAL VLSSNHSLTR LYIGENALGD SGVQVLCEKM KDPQCNLQKL GLVNSGLTSI CCSALTSVLK TNQNFTHLYL RSNALGDTGL RLLCEGLLHP DCKLQMLELD NCSLTSHSCW NLSTILTHNH SLRKLNLGNN DLGDLCVVTL CEVLKQQGCL LQSLQLGEMY LNRETKRALE ALQEEKPELT IVFEISW

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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) / Average electron dose: 53.46 e/Å2
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: -2.5 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

CTF correctionSoftware - Name: CTFFIND
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 59107
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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