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- EMDB-0476: Cryo-EM structure of NLRP3 bound to NEK7 -

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Basic information

Entry
Database: EMDB / ID: EMD-0476
TitleCryo-EM structure of NLRP3 bound to NEK7
Map data
Sample
  • Complex: NL3-NEK7
    • Complex: NLRP3
      • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
    • Complex: NEK7
      • Protein or peptide: Protein kinase R,Serine/threonine-protein kinase Nek7
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsInflammasome / Activator / Biological process Immunity / Inflammatory response / Innate immunity / Transcription / Transcription regulation Ligand / ATP binding / Nucleotide binding / IMMUNE SYSTEM
Function / homology
Function and homology information


NEK6-subfamily protein kinase / regulation of NLRP3 inflammasome complex assembly / Inhibition of PKR / small molecule sensor activity / detection of biotic stimulus / eukaryotic translation initiation factor 2alpha kinase activity / phosphatidylinositol phosphate binding / cysteine-type endopeptidase activator activity / response to interferon-alpha / positive regulation of stress-activated MAPK cascade ...NEK6-subfamily protein kinase / regulation of NLRP3 inflammasome complex assembly / Inhibition of PKR / small molecule sensor activity / detection of biotic stimulus / eukaryotic translation initiation factor 2alpha kinase activity / phosphatidylinositol phosphate binding / cysteine-type endopeptidase activator activity / response to interferon-alpha / positive regulation of stress-activated MAPK cascade / regulation of hematopoietic progenitor cell differentiation / positive regulation of T-helper 2 cell differentiation / negative regulation of osteoblast proliferation / Activation of NIMA Kinases NEK9, NEK6, NEK7 / interphase microtubule organizing center / NLRP3 inflammasome complex assembly / positive regulation of T-helper 2 cell cytokine production / cellular response to potassium ion / NLRP3 inflammasome complex / positive regulation of type 2 immune response / protein phosphatase regulator activity / Nuclear Pore Complex (NPC) Disassembly / osmosensory signaling pathway / peptidoglycan binding / SUMOylation of immune response proteins / regulation of hematopoietic stem cell proliferation / negative regulation of non-canonical NF-kappaB signal transduction / phosphatidylinositol-4-phosphate binding / regulation of hematopoietic stem cell differentiation / 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 viral genome replication / negative regulation of NF-kappaB transcription factor activity / pyroptosis / regulation of mitotic cell cycle / antiviral innate immune response / positive regulation of telomere capping / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / The NLRP3 inflammasome / protein maturation / negative regulation of acute inflammatory response / positive regulation of interleukin-4 production / Purinergic signaling in leishmaniasis infection / endoplasmic reticulum unfolded protein response / spindle assembly / EML4 and NUDC in mitotic spindle formation / positive regulation of chemokine production / signaling adaptor activity / positive regulation of telomerase activity / molecular condensate scaffold activity / positive regulation of telomere maintenance via telomerase / cellular response to amino acid starvation / positive regulation of interleukin-1 beta production / molecular function activator activity / ADP binding / positive regulation of cytokine production / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / non-specific protein-tyrosine kinase / response to virus / PKR-mediated signaling / non-membrane spanning protein tyrosine kinase activity / protein homooligomerization / defense response / Cytoprotection by HMOX1 / Metalloprotease DUBs / ISG15 antiviral mechanism / negative regulation of inflammatory response / cellular response to virus / positive regulation of inflammatory response / spindle pole / positive regulation of non-canonical NF-kappaB signal transduction / SARS-CoV-1 activates/modulates innate immune responses / Interferon alpha/beta signaling / double-stranded RNA binding / positive regulation of NF-kappaB transcription factor activity / kinase activity / defense response to virus / cellular response to lipopolysaccharide / DNA-binding transcription factor binding / microtubule / sequence-specific DNA binding / negative regulation of translation / positive regulation of MAPK cascade / protein autophosphorylation / molecular adaptor activity / ribosome / non-specific serine/threonine protein kinase / protein kinase activity / inflammatory response / translation / negative regulation of cell population proliferation / Golgi membrane / protein phosphorylation / protein serine kinase activity / innate immune response / protein serine/threonine kinase activity / centrosome / apoptotic process
Similarity search - Function
EIF2AK2, first double-stranded RNA binding domain / EIF2AK2, second double-stranded RNA binding 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 ...EIF2AK2, first double-stranded RNA binding domain / EIF2AK2, second double-stranded RNA binding 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. / Leucine rich repeat, ribonuclease inhibitor type / PAAD/DAPIN/Pyrin domain / NACHT nucleoside triphosphatase / PAAD/DAPIN/Pyrin domain / NACHT domain / NACHT-NTPase domain profile. / Leucine Rich repeat / Double-stranded RNA binding motif / Double-stranded RNA binding motif / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / 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
Interferon-induced, double-stranded RNA-activated protein kinase / 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: 3.8 Å
AuthorsSharif H / Wang L / Wu H
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)1R01 Al124491 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)1DP1 HD087988 United States
CitationJournal: Nature / Year: 2019
Title: Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome.
Authors: Humayun Sharif / Li Wang / Wei Li Wang / Venkat Giri Magupalli / Liudmila Andreeva / Qi Qiao / Arthur V Hauenstein / Zhaolong Wu / Gabriel Núñez / Youdong Mao / Hao Wu /
Abstract: The NLRP3 inflammasome can be activated by stimuli that include nigericin, uric acid crystals, amyloid-β fibrils and extracellular ATP. The mitotic kinase NEK7 licenses the assembly and activation ...The NLRP3 inflammasome can be activated by stimuli that include nigericin, uric acid crystals, amyloid-β fibrils and extracellular ATP. The mitotic kinase NEK7 licenses the assembly and activation of the NLRP3 inflammasome in interphase. Here we report a cryo-electron microscopy structure of inactive human NLRP3 in complex with NEK7, at a resolution of 3.8 Å. The earring-shaped NLRP3 consists of curved leucine-rich-repeat and globular NACHT domains, and the C-terminal lobe of NEK7 nestles against both NLRP3 domains. Structural recognition between NLRP3 and NEK7 is confirmed by mutagenesis both in vitro and in cells. Modelling of an active NLRP3-NEK7 conformation based on the NLRC4 inflammasome predicts an additional contact between an NLRP3-bound NEK7 and a neighbouring NLRP3. Mutations to this interface abolish the ability of NEK7 or NLRP3 to rescue NLRP3 activation in NEK7-knockout or NLRP3-knockout cells. These data suggest that NEK7 bridges adjacent NLRP3 subunits with bipartite interactions to mediate the activation of the NLRP3 inflammasome.
History
DepositionJan 18, 2019-
Header (metadata) releaseJun 19, 2019-
Map releaseJun 19, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00926
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.00926
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6npy
  • Surface level: 0.00926
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0476.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.00926 / Movie #1: 0.00926
Minimum - Maximum-0.022114016 - 0.04407179
Average (Standard dev.)0.00021862681 (±0.001666536)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 201.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z201.600201.600201.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0220.0440.000

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Supplemental data

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Sample components

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Entire : NL3-NEK7

EntireName: NL3-NEK7
Components
  • Complex: NL3-NEK7
    • Complex: NLRP3
      • Protein or peptide: NACHT, LRR and PYD domains-containing protein 3
    • Complex: NEK7
      • Protein or peptide: Protein kinase R,Serine/threonine-protein kinase Nek7
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: NL3-NEK7

SupramoleculeName: NL3-NEK7 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2

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Supramolecule #2: NLRP3

SupramoleculeName: NLRP3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: The sequence is according to NP_001230062.1 isoform which lacks 2 residues from N-terminus. Total residues are 1034aa as compared to 1036 in UNP Q96P20. There are mutations in YRKKYRKY ...Details: The sequence is according to NP_001230062.1 isoform which lacks 2 residues from N-terminus. Total residues are 1034aa as compared to 1036 in UNP Q96P20. There are mutations in YRKKYRKY instead its IYCAKYRAY mutations were intended to introduce so that the class of MBP and NLRP3 is avoided
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: NEK7

SupramoleculeName: NEK7 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #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: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 117.890984 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MASTRCKLAR YLEDLEDVDL KKFKMHLEDY PPQKGCIPLP RGQTEKADHV DLATLMIDFN GEEKAWAMAV WIFAAINRRD LYEKAKRDE PKWGSDNARV SNPTVICQED SIEEEWMGLL EYLSRISICK MKKIYCAKYR AYVRSRFQCI EDRNARLGES V SLNKRYTR ...String:
MASTRCKLAR YLEDLEDVDL KKFKMHLEDY PPQKGCIPLP RGQTEKADHV DLATLMIDFN GEEKAWAMAV WIFAAINRRD LYEKAKRDE PKWGSDNARV SNPTVICQED SIEEEWMGLL EYLSRISICK MKKIYCAKYR AYVRSRFQCI EDRNARLGES V SLNKRYTR LRLIKEHRSQ QEREQELLAI GKTKTCESPV SPIKMELLFD PDDEHSEPVH TVVFQGAAGI GKTILARKMM LD WASGTLY QDRFDYLFYI HCREVSLVTQ RSLGDLIMSC CPDPNPPIHK IVRKPSRILF LMDGFDELQG AFDEHIGPLC TDW QKAERG DILLSSLIRK KLLPEASLLI TTRPVALEKL QHLLDHPRHV EILGFSEAKR KEYFFKYFSD EAQARAAFSL IQEN EVLFT MCFIPLVCWI VCTGLKQQME SGKSLAQTSK TTTAVYVFFL SSLLQPRGGS QEHGLCAHLW GLCSLAADGI WNQKI LFEE SDLRNHGLQK ADVSAFLRMN LFQKEVDCEK FYSFIHMTFQ EFFAAMYYLL EEEKEGRTNV PGSRLKLPSR DVTVLL ENY GKFEKGYLIF VVRFLFGLVN QERTSYLEKK LSCKISQQIR LELLKWIEVK AKAKKLQIQP SQLELFYCLY EMQEEDF VQ RAMDYFPKIE INLSTRMDHM VSSFCIENCH RVESLSLGFL HNMPKEEEEE EKEGRHLDMV QCVLPSSSHA ACSHGLVN S HLTSSFCRGL FSVLSTSQSL TELDLSDNSL GDPGMRVLCE TLQHPGCNIR RLWLGRCGLS HECCFDISLV LSSNQKLVE LDLSDNALGD FGIRLLCVGL KHLLCNLKKL WLVSCCLTSA CCQDLASVLS TSHSLTRLYV GENALGDSGV AILCEKAKNP QCNLQKLGL VNSGLTSVCC SALSSVLSTN QNLTHLYLRG NTLGDKGIKL LCEGLLHPDC KLQVLELDNC NLTSHCCWDL S TLLTSSQS LRKLSLGNND LGDLGVMMFC EVLKQQSCLL QNLGLSEMYF NYETKSALET LQEEKPELTV VFEPSW

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

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Macromolecule #2: Protein kinase R,Serine/threonine-protein kinase Nek7

MacromoleculeName: Protein kinase R,Serine/threonine-protein kinase Nek7 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.01515 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSTDKRFGMD FRIEKKIGRG QFSEVYRAAC RLDGKTVALK KVQIFDLMDA KARADCIKEI DLLAQLNHPN VIKYYVCFIT GNELNIVLE LADAGDLSRM IKHFKKQKRL IPERTVWKYF VQLCSALEHM HSRRVMHRDI KPANVFITAT GVVKLGDLGL G RFFSSKTT ...String:
GSTDKRFGMD FRIEKKIGRG QFSEVYRAAC RLDGKTVALK KVQIFDLMDA KARADCIKEI DLLAQLNHPN VIKYYVCFIT GNELNIVLE LADAGDLSRM IKHFKKQKRL IPERTVWKYF VQLCSALEHM HSRRVMHRDI KPANVFITAT GVVKLGDLGL G RFFSSKTT AAHSLVGTPY YMSPERIHEN GYNFKSDIWS LGCLLYEMAA LQSPFYGDKM NLYSLCKKIE QCDYPPLPSD HY SEELRQL VNMCINPDPE KRPDVTYVYD VAKRMHACTA SS

UniProtKB: Interferon-induced, double-stranded RNA-activated protein kinase, Serine/threonine-protein kinase Nek7

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.1 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 108771
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6npy:
Cryo-EM structure of NLRP3 bound to NEK7

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