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

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Basic information

Entry
Database: PDB / ID: 7lfh
TitleCryo-EM structure of NLRP3 double-ring cage, 6-fold (12-mer)
ComponentsNACHT, LRR and PYD domains-containing protein 3
KeywordsIMMUNE SYSTEM / NLRP3 / NEK7 / nucleotid-binding / ATP-binding / inflammasome / immunity / innate immunity / NACHT / LRR / PYD
Function / homology
Function and homology information


canonical inflammasome complex / positive regulation of T-helper 17 cell differentiation / The NLRP3 inflammasome / detection of biotic stimulus / Metalloprotease DUBs / molecular sensor activity / positive regulation of type 2 immune response / phosphatidylinositol phosphate binding / acute inflammatory response / positive regulation of T-helper 2 cell differentiation ...canonical inflammasome complex / positive regulation of T-helper 17 cell differentiation / The NLRP3 inflammasome / detection of biotic stimulus / Metalloprotease DUBs / molecular sensor activity / positive regulation of type 2 immune response / phosphatidylinositol phosphate binding / acute inflammatory response / positive regulation of T-helper 2 cell differentiation / positive regulation of T-helper 2 cell cytokine production / positive regulation of interleukin-18 production / interphase microtubule organizing center / cellular response to peptidoglycan / positive regulation of cytokine production involved in immune response / NLRP3 inflammasome complex / positive regulation of interleukin-5 production / positive regulation of interleukin-13 production / interleukin-18-mediated signaling pathway / NLRP3 inflammasome complex assembly / cysteine-type endopeptidase activator activity / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / osmosensory signaling pathway / interleukin-1-mediated signaling pathway / negative regulation of interleukin-1 beta production / pattern recognition receptor signaling pathway / positive regulation of interleukin-4 production / negative regulation of acute inflammatory response / protein secretion / microtubule organizing center / positive regulation of NLRP3 inflammasome complex assembly / pyroptotic inflammatory response / signaling adaptor activity / positive regulation of interleukin-1 beta production / cellular response to virus / protein maturation / molecular condensate scaffold activity / negative regulation of inflammatory response / positive regulation of non-canonical NF-kappaB signal transduction / 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 / molecular adaptor activity / response to ethanol / protein-macromolecule adaptor activity / 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 / DNA-templated transcription / extracellular region / ATP binding / membrane / identical protein binding / nucleus / 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 profile. / 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 profile. / NACHT nucleoside triphosphatase / NACHT domain / NACHT-NTPase domain profile. / DAPIN domain / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsAndreeva, L. / Rawson, S. / Wu, H.
Funding support United States, 3items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 15, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 5, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 2.0Apr 27, 2022Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Refinement description / Structure summary
Category: atom_site / em_software ...atom_site / em_software / pdbx_contact_author / pdbx_struct_sheet_hbond / pdbx_validate_close_contact / pdbx_validate_peptide_omega / pdbx_validate_torsion / refine_ls_restr / software / struct_conf / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.pdbx_formal_charge / _em_software.category / _struct_sheet_range.id / _struct_sheet_range.sheet_id
Description: Polymer backbone linkage / Provider: author / Type: Coordinate replacement
Revision 2.1May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: NACHT, LRR and PYD domains-containing protein 3
B: NACHT, LRR and PYD domains-containing protein 3
C: NACHT, LRR and PYD domains-containing protein 3
D: NACHT, LRR and PYD domains-containing protein 3
E: NACHT, LRR and PYD domains-containing protein 3
F: NACHT, LRR and PYD domains-containing protein 3
G: NACHT, LRR and PYD domains-containing protein 3
H: NACHT, LRR and PYD domains-containing protein 3
I: NACHT, LRR and PYD domains-containing protein 3
J: NACHT, LRR and PYD domains-containing protein 3
K: NACHT, LRR and PYD domains-containing protein 3
L: NACHT, LRR and PYD domains-containing protein 3


Theoretical massNumber of molelcules
Total (without water)1,425,62712
Polymers1,425,62712
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
NACHT, LRR and PYD domains-containing protein 3 / Cold autoinflammatory syndrome 1 protein homolog / Cryopyrin / Mast cell maturation-associated- ...Cold autoinflammatory syndrome 1 protein homolog / Cryopyrin / Mast cell maturation-associated-inducible protein 1 / PYRIN-containing APAF1-like protein 1


Mass: 118802.219 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Nlrp3, Cias1, Mmig1, Nalp3, Pypaf1 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q8R4B8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: NLRP3 double-ring cage, 6-fold (12-mer) / Type: COMPLEX / Details: NLRP3 oligomer / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.42 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T
Buffer solutionpH: 7.5
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: -2400 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.51 sec. / Electron dose: 53.225 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6800
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.

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.8.5image acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122941 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6NPY

6npy


Pdb chain-ID: A / Accession code: 6NPY / Pdb chain residue range: 131-1036 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00478324
ELECTRON MICROSCOPYf_angle_d0.941105672
ELECTRON MICROSCOPYf_dihedral_angle_d6.68110284
ELECTRON MICROSCOPYf_chiral_restr0.05112144
ELECTRON MICROSCOPYf_plane_restr0.00513284

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