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- PDB-6nt9: Cryo-EM structure of the complex between human TBK1 and chicken STING -

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Basic information

Entry
Database: PDB / ID: 6nt9
TitleCryo-EM structure of the complex between human TBK1 and chicken STING
Components
  • Serine/threonine-protein kinase TBK1
  • Stimulator of interferon genes protein
KeywordsIMMUNE SYSTEM / ER / kinase / adaptor
Function / homology
Function and homology information


STING mediated induction of host immune responses / IRF3 mediated activation of type 1 IFN / STAT6-mediated induction of chemokines / positive regulation of xenophagy / serine/threonine protein kinase complex / proton channel activity / 2',3'-cyclic GMP-AMP binding / regulation of type I interferon production / dendritic cell proliferation / cyclic-di-GMP binding ...STING mediated induction of host immune responses / IRF3 mediated activation of type 1 IFN / STAT6-mediated induction of chemokines / positive regulation of xenophagy / serine/threonine protein kinase complex / proton channel activity / 2',3'-cyclic GMP-AMP binding / regulation of type I interferon production / dendritic cell proliferation / cyclic-di-GMP binding / cGAS/STING signaling pathway / IRF3-mediated induction of type I IFN / positive regulation of type I interferon-mediated signaling pathway / reticulophagy / Interleukin-37 signaling / TNFR1-induced proapoptotic signaling / TRAF6 mediated IRF7 activation / type I interferon-mediated signaling pathway / cytoplasmic pattern recognition receptor signaling pathway / autophagosome membrane / positive regulation of interferon-alpha production / antiviral innate immune response / positive regulation of macroautophagy / autophagosome assembly / autophagosome / positive regulation of type I interferon production / canonical NF-kappaB signal transduction / positive regulation of autophagy / negative regulation of TORC1 signaling / TICAM1-dependent activation of IRF3/IRF7 / positive regulation of TORC1 signaling / Regulation of innate immune responses to cytosolic DNA / positive regulation of interferon-beta production / activation of innate immune response / endoplasmic reticulum-Golgi intermediate compartment membrane / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Negative regulators of DDX58/IFIH1 signaling / Neutrophil degranulation / Regulation of TNFR1 signaling / phosphoprotein binding / peptidyl-threonine phosphorylation / response to virus / DDX58/IFIH1-mediated induction of interferon-alpha/beta / SARS-CoV-1 activates/modulates innate immune responses / protein complex oligomerization / positive regulation of peptidyl-serine phosphorylation / TRAF3-dependent IRF activation pathway / cytoplasmic vesicle / protein phosphatase binding / defense response to virus / peptidyl-serine phosphorylation / positive regulation of canonical NF-kappaB signal transduction / Potential therapeutics for SARS / nucleic acid binding / non-specific serine/threonine protein kinase / protein kinase activity / defense response to Gram-positive bacterium / inflammatory response / Golgi membrane / negative regulation of gene expression / protein phosphorylation / protein serine kinase activity / innate immune response / intracellular membrane-bounded organelle / protein serine/threonine kinase activity / endoplasmic reticulum membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / perinuclear region of cytoplasm / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / nucleoplasm / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Substrate Binding Domain Of Dnak; Chain:A; Domain 2 - #420 / TANK binding kinase 1, ubiquitin-like domain / TANK-binding kinase 1, coiled-coil domain 1 / TANK-binding kinase 1 coiled-coil domain 1 / TANK binding kinase 1 ubiquitin-like domain / : / Stimulator of interferon genes protein / Stimulator of interferon genes protein, C-terminal domain superfamily / Transmembrane protein 173 / Substrate Binding Domain Of Dnak; Chain:A; Domain 2 ...Substrate Binding Domain Of Dnak; Chain:A; Domain 2 - #420 / TANK binding kinase 1, ubiquitin-like domain / TANK-binding kinase 1, coiled-coil domain 1 / TANK-binding kinase 1 coiled-coil domain 1 / TANK binding kinase 1 ubiquitin-like domain / : / Stimulator of interferon genes protein / Stimulator of interferon genes protein, C-terminal domain superfamily / Transmembrane protein 173 / Substrate Binding Domain Of Dnak; Chain:A; Domain 2 / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Roll / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Up-down Bundle / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Stimulator of interferon genes protein / Stimulator of interferon genes protein / Serine/threonine-protein kinase TBK1
Similarity search - Component
Biological speciesHomo sapiens (human)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsShang, G. / Zhang, C. / Chen, Z.J. / Bai, X. / Zhang, X.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM088197 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130289 United States
Howard Hughes Medical Institute (HHMI) United States
Welch FoundationI-1389 United States
Welch FoundationI-1702 United States
Welch FoundationI-1944 United States
CitationJournal: Nature / Year: 2019
Title: Structural basis of STING binding with and phosphorylation by TBK1.
Authors: Conggang Zhang / Guijun Shang / Xiang Gui / Xuewu Zhang / Xiao-Chen Bai / Zhijian J Chen /
Abstract: The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system. Cyclic ...The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system. Cyclic GMP-AMP synthase (cGAS) is a sensor of cytoplasmic DNA that activates the type-I interferon pathway. On binding to DNA, cGAS is activated to catalyse the synthesis of cyclic GMP-AMP (cGAMP) from GTP and ATP. cGAMP functions as a second messenger that binds to and activates stimulator of interferon genes (STING). STING then recruits and activates tank-binding kinase 1 (TBK1), which phosphorylates STING and the transcription factor IRF3 to induce type-I interferons and other cytokines. However, how cGAMP-bound STING activates TBK1 and IRF3 is not understood. Here we present the cryo-electron microscopy structure of human TBK1 in complex with cGAMP-bound, full-length chicken STING. The structure reveals that the C-terminal tail of STING adopts a β-strand-like conformation and inserts into a groove between the kinase domain of one TBK1 subunit and the scaffold and dimerization domain of the second subunit in the TBK1 dimer. In this binding mode, the phosphorylation site Ser366 in the STING tail cannot reach the kinase-domain active site of bound TBK1, which suggests that STING phosphorylation by TBK1 requires the oligomerization of both proteins. Mutational analyses validate the interaction mode between TBK1 and STING and support a model in which high-order oligomerization of STING and TBK1, induced by cGAMP, leads to STING phosphorylation by TBK1.
History
DepositionJan 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 3, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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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Assembly

Deposited unit
A: Serine/threonine-protein kinase TBK1
C: Stimulator of interferon genes protein
B: Serine/threonine-protein kinase TBK1
D: Stimulator of interferon genes protein


Theoretical massNumber of molelcules
Total (without water)259,0484
Polymers259,0484
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Serine/threonine-protein kinase TBK1 / NF-kappa-B-activating kinase / T2K / TANK-binding kinase 1


Mass: 85316.695 Da / Num. of mol.: 2 / Mutation: D135N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TBK1, NAK / Production host: Homo sapiens (human)
References: UniProt: Q9UHD2, non-specific serine/threonine protein kinase
#2: Protein Stimulator of interferon genes protein /


Mass: 44207.070 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: TMEM173 / Production host: Homo sapiens (human) / References: UniProt: A0A1D5P7Q9, UniProt: E1C7U0*PLUS

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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-IDSource
1full-length chicken STING and human TBK1COMPLEXall0RECOMBINANT
2STINGCOMPLEX#21RECOMBINANT
3TBK1COMPLEX#11RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Gallus gallus (chicken)9031
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606HEK293 GnTI-
23Homo sapiens (human)9606HEK293 GnTI-
Buffer solutionpH: 8
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 48 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86276 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 4IM0

4im0


Accession code: 4IM0 / Source name: PDB / Type: experimental model

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