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- EMDB-13727: Cryo-EM structure of the NLRP3 PYD filament -

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Basic information

Entry
Database: EMDB / ID: EMD-13727
TitleCryo-EM structure of the NLRP3 PYD filament
Map data
Sample
  • Organelle or cellular component: filament formed by the human NLRP3 Pyrin domain
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
KeywordsNLRP3 / Pyrin domain / filament / IMMUNE SYSTEM
Function / homology
Function and homology information


small molecule sensor activity / detection of biotic stimulus / cysteine-type endopeptidase activator activity / phosphatidylinositol phosphate binding / positive regulation of T-helper 2 cell differentiation / NLRP3 inflammasome complex assembly / interphase microtubule organizing center / positive regulation of T-helper 2 cell cytokine production / positive regulation of type 2 immune response / NLRP3 inflammasome complex ...small molecule sensor activity / detection of biotic stimulus / cysteine-type endopeptidase activator activity / phosphatidylinositol phosphate binding / positive regulation of T-helper 2 cell differentiation / NLRP3 inflammasome complex assembly / interphase microtubule organizing center / positive regulation of T-helper 2 cell cytokine production / positive regulation of type 2 immune response / NLRP3 inflammasome complex / peptidoglycan binding / osmosensory signaling pathway / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / pattern recognition receptor signaling pathway / negative regulation of interleukin-1 beta production / 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 / The NLRP3 inflammasome / protein maturation / Purinergic signaling in leishmaniasis infection / signaling adaptor activity / molecular condensate scaffold 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 / Cytoprotection by HMOX1 / Metalloprotease DUBs / cellular response to virus / ADP binding / defense response / negative regulation of inflammatory response / positive regulation of inflammatory response / positive regulation of non-canonical NF-kappaB signal transduction / SARS-CoV-1 activates/modulates innate immune responses / protein-macromolecule adaptor activity / positive regulation of NF-kappaB transcription factor activity / cellular response to lipopolysaccharide / DNA-binding transcription factor binding / sequence-specific DNA binding / molecular adaptor activity / inflammatory response / Golgi membrane / innate immune response / apoptotic process / SARS-CoV-2 activates/modulates innate and adaptive immune responses / endoplasmic reticulum / signal transduction / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular region / ATP binding / identical protein 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 / 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 speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHochheiser IV / Hagelueken G
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)EXC2151 - 390873048 Germany
CitationJournal: Sci Adv / Year: 2022
Title: Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation.
Authors: Inga V Hochheiser / Heide Behrmann / Gregor Hagelueken / Juan F Rodríguez-Alcázar / Anja Kopp / Eicke Latz / Elmar Behrmann / Matthias Geyer /
Abstract: Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide- ...Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)-like receptors with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) mediate oligomerization into filamentous assemblies. We describe the cryo-electron microscopy (cryo-EM) structure of the human NLRP3 filament and identify a pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends designated as A- and B-ends. Coupling a titration polymerization assay to cryo-EM, we demonstrate that ASC adaptor protein elongation on NLRP3 nucleation seeds is unidirectional, associating exclusively to the B-end of the filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing a continuous transition between NLRP3 and ASC. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3, ASC, and Caspase-1 proteins.
History
DepositionOct 12, 2021-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13727.png

Downloads & links

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Map

FileDownload / File: emd_13727.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 220 pix.
= 234.74 Å		1.07 Å/pix.
x 220 pix.
= 234.74 Å		1.07 Å/pix.
x 220 pix.
= 234.74 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.123
Minimum - Maximum-0.39900687 - 0.62916315
Average (Standard dev.)0.0011606073 (±0.0303657)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 234.74 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : filament formed by the human NLRP3 Pyrin domain

EntireName: filament formed by the human NLRP3 Pyrin domain
Components
  • Organelle or cellular component: filament formed by the human NLRP3 Pyrin domain
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3

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Supramolecule #1: filament formed by the human NLRP3 Pyrin domain

SupramoleculeName: filament formed by the human NLRP3 Pyrin domain / type: organelle_or_cellular_component / 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 3

MacromoleculeName: NACHT, LRR and PYD domains-containing protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.486243 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MASTRCKLAR YLEDLEDVDL KKFKMHLEDY PPQKGCIPLP RGQTEKADHV DLATLMIDFN GEEKAWAMAV WIFAAINRRD LYEKAKRDE PKWGSDNARV SNPTVISQE

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

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 293 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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: 14.32 Å
Applied symmetry - Helical parameters - Δ&Phi: 54.88 °
Applied symmetry - Helical parameters - Axial symmetry: C3 (3 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 60333
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3qf2

Final angle assignmentType: NOT APPLICABLE

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