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- EMDB-28175: Cryo-EM structure of the active NLRP3 inflammasome disk -

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Basic information

Entry
Database: EMDB / ID: EMD-28175
TitleCryo-EM structure of the active NLRP3 inflammasome disk
Map dataPrimary Map
Sample
  • Complex: a complex of NLRP3 and NEK7
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
    • Protein or peptide: Serine/threonine-protein kinase Nek7
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


NEK6-subfamily protein kinase / small molecule sensor activity / detection of biotic stimulus / phosphatidylinositol phosphate binding / cysteine-type endopeptidase activator activity / positive regulation of T-helper 2 cell differentiation / Activation of NIMA Kinases NEK9, NEK6, NEK7 / interphase microtubule organizing center / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production ...NEK6-subfamily protein kinase / small molecule sensor activity / detection of biotic stimulus / phosphatidylinositol phosphate binding / cysteine-type endopeptidase activator activity / positive regulation of T-helper 2 cell differentiation / Activation of NIMA Kinases NEK9, NEK6, NEK7 / interphase microtubule organizing center / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production / NLRP3 inflammasome complex / positive regulation of type 2 immune response / cellular response to potassium ion / Nuclear Pore Complex (NPC) Disassembly / osmosensory signaling pathway / peptidoglycan binding / phosphatidylinositol-4-phosphate binding / negative regulation of non-canonical NF-kappaB signal transduction / microtubule organizing center / negative regulation of interleukin-1 beta production / pattern recognition receptor signaling pathway / positive regulation of NLRP3 inflammasome complex assembly / negative regulation of NF-kappaB transcription factor activity / pyroptotic inflammatory response / positive regulation of telomere capping / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / protein maturation / The NLRP3 inflammasome / negative regulation of acute inflammatory response / positive regulation of interleukin-4 production / spindle assembly / Purinergic signaling in leishmaniasis infection / signaling adaptor activity / EML4 and NUDC in mitotic spindle formation / positive regulation of telomerase activity / regulation of mitotic cell cycle / positive regulation of telomere maintenance via telomerase / molecular condensate scaffold activity / positive regulation of interleukin-1 beta production / molecular function activator activity / ADP binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / defense response / protein homooligomerization / Cytoprotection by HMOX1 / Metalloprotease DUBs / cellular response to virus / spindle pole / 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 / positive regulation of NF-kappaB transcription factor activity / DNA-binding transcription factor binding / cellular response to lipopolysaccharide / microtubule / sequence-specific DNA binding / molecular adaptor activity / inflammatory response / Golgi membrane / protein phosphorylation / protein serine kinase activity / innate immune response / protein serine/threonine kinase activity / centrosome / 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 / nucleoplasm / ATP binding / membrane / identical protein binding / metal ion 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 / 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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Serine/threonine-protein kinase Nek7 / NACHT, LRR and PYD domains-containing protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHao W / Le X
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nature / Year: 2023
Title: Cryo-EM structures of the active NLRP3 inflammasome disc.
Authors: Le Xiao / Venkat Giri Magupalli / Hao Wu /
Abstract: Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers, and NACHT-, leucine-rich repeat (LRR)- and pyrin domain (PYD)- ...Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers, and NACHT-, leucine-rich repeat (LRR)- and pyrin domain (PYD)-containing protein  3 (NLRP3) is an inflammasome sensor of membrane damage highly important in regard to the induction of inflammation. Here we report cryogenic electron microscopy structures of disc-shaped active NLRP3 oligomers in complex with adenosine 5'-O-(3-thio)triphosphate, the centrosomal NIMA-related kinase 7 (NEK7) and the adaptor protein ASC, which recruits caspase-1. In these NLRP3-NEK7-ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by about 85° relative to the ADP-bound inactive conformation. The fish-specific NACHT-associated domain conserved in NLRP3 but absent in most NLRPs becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disc. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all NLRP3 subunits combine to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disc interfaces. Together with previous structures of an inactive NLRP3 cage in which LRR-LRR interactions play an important role, we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disc.
History
DepositionSep 16, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28175.png

Downloads & links

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Map

FileDownload / File: emd_28175.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary Map
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.4964914 - 2.5688908
Average (Standard dev.)0.0042392532 (±0.04771799)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 542.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_28175_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_28175_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : a complex of NLRP3 and NEK7

EntireName: a complex of NLRP3 and NEK7
Components
  • Complex: a complex of NLRP3 and NEK7
    • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
    • Protein or peptide: Serine/threonine-protein kinase Nek7
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: a complex of NLRP3 and NEK7

SupramoleculeName: a complex of NLRP3 and NEK7 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
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: 10 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 102.67557 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KKDYRKKYRK YVRSRFQCIE DRNARLGESV SLNKRYTRLR LIKEHRSQQE REQELLAIGK TKTCESPVSP IKMELLFDPD DEHSEPVHT VVFQGAAGIG KTILARKMML DWASGTLYQD RFDYLFYIHC REVSLVTQRS LGDLIMSCCP DPNPPIHKIV R KPSRILFL ...String:
KKDYRKKYRK YVRSRFQCIE DRNARLGESV SLNKRYTRLR LIKEHRSQQE REQELLAIGK TKTCESPVSP IKMELLFDPD DEHSEPVHT VVFQGAAGIG KTILARKMML DWASGTLYQD RFDYLFYIHC REVSLVTQRS LGDLIMSCCP DPNPPIHKIV R KPSRILFL MDGFDELQGA FDEHIGPLCT DWQKAERGDI LLSSLIRKKL LPEASLLITT RPVALEKLQH LLDHPRHVEI LG FSEAKRK EYFFKYFSDE AQARAAFSLI QENEVLFTMC FIPLVCWIVC TGLKQQMESG KSLAQTSKTT TAVYVFFLSS LLQ PRGGSQ EHGLCAHLWG LCSLAADGIW NQKILFEESD LRNHGLQKAD VSAFLRMNLF QKEVDCEKFY SFIHMTFQEF FAAM YYLLE EEKEGRTNVP GSRLKLPSRD VTVLLENYGK FEKGYLIFVV RFLFGLVNQE RTSYLEKKLS CKISQQIRLE LLKWI EVKA KAKKLQIQPS QLELFYCLYE MQEEDFVQRA MDYFPKIEIN LSTRMDHMVS SFCIENCHRV ESLSLGFLHN MPKEEE EEE KEGRHLDMVQ CVLPSSSHAA CSHGLVNSHL TSSFCRGLFS VLSTSQSLTE LDLSDNSLGD PGMRVLCETL QHPGCNI RR LWLGRCGLSH ECCFDISLVL SSNQKLVELD LSDNALGDFG IRLLCVGLKH LLCNLKKLWL VSCCLTSACC QDLASVLS T SHSLTRLYVG ENALGDSGVA ILCEKAKNPQ CNLQKLGLVN SGLTSVCCSA LSSVLSTNQN LTHLYLRGNT LGDKGIKLL CEGLLHPDCK LQVLELDNCN LTSHCCWDLS TLLTSSQSLR KLSLGNNDLG DLGVMMFCEV LKQQSCLLQN LGLSEMYFNY ETKSALETL QEEKPELTVV FEP

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Macromolecule #2: Serine/threonine-protein kinase Nek7

MacromoleculeName: Serine/threonine-protein kinase Nek7 / type: protein_or_peptide / ID: 2 / Number of copies: 10 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.038203 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KALRPDMGYN TLANFRIEKK IGRGQFSEVY RAACLLDGVP VALKKVQIFD LMDAKARADC IKEIDLLKQL NHPNVIKYYA SFIEDNELN IVLELADAGD LSRMIKHFKK QKRLIPERTV WKYFVQLCSA LEHMHSRRVM HRDIKPANVF ITATGVVKLG D LGLGRFFS ...String:
KALRPDMGYN TLANFRIEKK IGRGQFSEVY RAACLLDGVP VALKKVQIFD LMDAKARADC IKEIDLLKQL NHPNVIKYYA SFIEDNELN IVLELADAGD LSRMIKHFKK QKRLIPERTV WKYFVQLCSA LEHMHSRRVM HRDIKPANVF ITATGVVKLG D LGLGRFFS SKTTAAHSLV GTPYYMSPER IHENGYNFKS DIWSLGCLLY EMAALQSPFY GDKMNLYSLC KKIEQCDYPP LP SDHYSEE LRQLVNMCIN PDPEKRPDVT YVYDVAKRMH A

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 10 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 10 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: COMMON LINE
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51576

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