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

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Basic information

Entry
Database: PDB / ID: 6npy
TitleCryo-EM structure of NLRP3 bound to NEK7
Components
  • NACHT, LRR and PYD domains-containing protein 3
  • Protein kinase R,Serine/threonine-protein kinase Nek7
KeywordsIMMUNE SYSTEM / Inflammasome / Activator / Biological process Immunity / Inflammatory response / Innate immunity / Transcription / Transcription regulation Ligand / ATP binding / Nucleotide binding
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 / Activation of NIMA Kinases NEK9, NEK6, NEK7 / negative regulation of osteoblast proliferation / 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 / 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 / endoplasmic reticulum unfolded protein response / EML4 and NUDC in mitotic spindle formation / positive regulation of chemokine production / 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 / non-membrane spanning protein tyrosine kinase activity / PKR-mediated signaling / 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 / DNA-binding transcription factor binding / cellular response to lipopolysaccharide / microtubule / negative regulation of translation / sequence-specific DNA binding / positive regulation of MAPK cascade / protein autophosphorylation / molecular adaptor activity / non-specific serine/threonine protein kinase / ribosome / protein kinase activity / inflammatory response / translation / negative regulation of cell population proliferation / Golgi membrane / protein phosphorylation / innate immune response / protein serine kinase activity / centrosome / protein serine/threonine kinase activity / 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 / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSharif, H. / Wang, L. / Wang, W.L. / Wu, H.
Funding support United States, 2items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 3, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Aug 14, 2019Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / entity ...atom_site / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / struct_conf / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet_range
Item: _atom_site.label_seq_id / _entity.formula_weight ..._atom_site.label_seq_id / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_mutation / _entity_name_com.name / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_seq_id / _struct_conf.beg_label_seq_id / _struct_conf.end_label_seq_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_label_seq_id
Revision 2.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: NACHT, LRR and PYD domains-containing protein 3
B: Protein kinase R,Serine/threonine-protein kinase Nek7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,3333
Polymers149,9062
Non-polymers4271
Water0
1


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

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Components

#1: Protein NACHT, LRR and PYD domains-containing protein 3 / Angiotensin/vasopressin receptor AII/AVP-like / Caterpiller protein 1.1 / CLR1.1 / Cold-induced ...Angiotensin/vasopressin receptor AII/AVP-like / Caterpiller protein 1.1 / CLR1.1 / Cold-induced autoinflammatory syndrome 1 protein / Cryopyrin / PYRIN-containing APAF1-like protein 1


Mass: 117890.984 Da / Num. of mol.: 1 / Mutation: D133I, R135C, K136A, K140A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NLRP3, C1orf7, CIAS1, NALP3, PYPAF1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q96P20
#2: Protein Protein kinase R,Serine/threonine-protein kinase Nek7 / Eukaryotic translation initiation factor 2-alpha kinase 2 / eIF-2A protein kinase 2 / Interferon- ...Eukaryotic translation initiation factor 2-alpha kinase 2 / eIF-2A protein kinase 2 / Interferon-inducible RNA-dependent protein kinase / P1/eIF-2A protein kinase / Protein kinase RNA-activated / Protein kinase R / Tyrosine-protein kinase EIF2AK2 / p68 kinase / Never in mitosis A-related kinase 7 / NimA-related protein kinase 7


Mass: 32015.150 Da / Num. of mol.: 1 / Mutation: L54R, V58K, P59T, K87A, A99V, S100C, E103T, D104G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EIF2AK2, PKR, PRKR, NEK7 / Production host: Escherichia coli (E. coli)
References: UniProt: P19525, UniProt: Q8TDX7, non-specific serine/threonine protein kinase
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
Sequence detailsThe sequence is according to NP_001230062.1 isoform which lacks 2 residues from N-terminus. Total ...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.

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1NL3-NEK7COMPLEX#20RECOMBINANT
2NLRP3COMPLEX#11RECOMBINANTThe 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
3NEK7COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.1 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -3000 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108771 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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