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- EMDB-32851: CT-mut (D523K,D524K,E527K) TLR3-poly(I:C) complex -

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Basic information

Entry
Database: EMDB / ID: EMD-32851
TitleCT-mut (D523K,D524K,E527K) TLR3-poly(I:C) complex
Map dataCT-mut mutant (D523K,D524K,E527K) TLR3 ectodomain
Sample
  • Complex: CT-mut(D523K,D524K,E527K) TLR3 ectodomain-poly I:C complex
    • Complex: CT-mut(D523K,D524K,E527K) TLR3 ectodomain
      • Protein or peptide: Toll-like receptor 3
    • Complex: poly(I:C)
      • RNA: RNA (46-MER)
      • RNA: RNA (46-MER)
Function / homology
Function and homology information


TLR3 deficiency - HSE / UNC93B1 deficiency - HSE / TICAM1 deficiency - HSE / type III interferon production / positive regulation of type III interferon production / response to dsRNA / TRAF3 deficiency - HSE / regulation of dendritic cell cytokine production / Toll Like Receptor 3 (TLR3) Cascade / TLR3-mediated TICAM1-dependent programmed cell death ...TLR3 deficiency - HSE / UNC93B1 deficiency - HSE / TICAM1 deficiency - HSE / type III interferon production / positive regulation of type III interferon production / response to dsRNA / TRAF3 deficiency - HSE / regulation of dendritic cell cytokine production / Toll Like Receptor 3 (TLR3) Cascade / TLR3-mediated TICAM1-dependent programmed cell death / I-kappaB phosphorylation / inflammatory response to wounding / toll-like receptor 3 signaling pathway / necroptotic signaling pathway / detection of virus / activation of NF-kappaB-inducing kinase activity / RIP-mediated NFkB activation via ZBP1 / positive regulation of cytokine production involved in inflammatory response / endolysosome membrane / hyperosmotic response / Trafficking and processing of endosomal TLR / positive regulation of macrophage cytokine production / pattern recognition receptor activity / toll-like receptor signaling pathway / response to exogenous dsRNA / cellular response to exogenous dsRNA / negative regulation of osteoclast differentiation / positive regulation of interferon-alpha production / cellular response to interferon-beta / positive regulation of chemokine production / extrinsic apoptotic signaling pathway / JNK cascade / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / positive regulation of interleukin-12 production / positive regulation of interferon-beta production / TICAM1, RIP1-mediated IKK complex recruitment / extracellular matrix / positive regulation of interleukin-8 production / microglial cell activation / positive regulation of JNK cascade / cellular response to virus / positive regulation of inflammatory response / cellular response to type II interferon / positive regulation of interleukin-6 production / positive regulation of non-canonical NF-kappaB signal transduction / cellular response to mechanical stimulus / male gonad development / positive regulation of angiogenesis / transmembrane signaling receptor activity / positive regulation of type II interferon production / cellular response to xenobiotic stimulus / double-stranded RNA binding / positive regulation of tumor necrosis factor production / signaling receptor activity / defense response to virus / positive regulation of canonical NF-kappaB signal transduction / early endosome / endosome membrane / defense response to bacterium / positive regulation of apoptotic process / lysosomal membrane / Golgi membrane / innate immune response / endoplasmic reticulum membrane / positive regulation of gene expression / signal transduction / positive regulation of transcription by RNA polymerase II / extracellular space / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Toll-like receptor 3 trans-membrane domain / Toll-like receptor 3 trans-membrane domain / Toll-like receptor / Leucine Rich repeat / TIR domain / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance / Leucine-rich repeat, SDS22-like subfamily / TIR domain profile. ...Toll-like receptor 3 trans-membrane domain / Toll-like receptor 3 trans-membrane domain / Toll-like receptor / Leucine Rich repeat / TIR domain / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance / Leucine-rich repeat, SDS22-like subfamily / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Toll-like receptor 3
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsLim CS / Jang YH / Lee GY / Han GM / Lee JO
Funding support Korea, Republic Of, 2 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2019M3E5D6066058 Korea, Republic Of
National Research Foundation (NRF, Korea)2017M3A9F6029753 Korea, Republic Of
CitationJournal: Nat Commun / Year: 2022
Title: TLR3 forms a highly organized cluster when bound to a poly(I:C) RNA ligand.
Authors: Chan Seok Lim / Yoon Ha Jang / Ga Young Lee / Gu Min Han / Hye Jin Jeong / Ji Won Kim / Jie-Oh Lee /
Abstract: Toll-like Receptor 3 (TLR3) initiates a potent anti-viral immune response by binding to double-stranded RNA ligands. Previous crystallographic studies showed that TLR3 forms a homodimer when bound to ...Toll-like Receptor 3 (TLR3) initiates a potent anti-viral immune response by binding to double-stranded RNA ligands. Previous crystallographic studies showed that TLR3 forms a homodimer when bound to a 46-base pair RNA ligand. However, this short RNA fails to initiate a robust immune response. To obtain structural insights into the length dependency of TLR3 ligands, we determine the cryo-electron microscopy structure of full-length TLR3 in a complex with a synthetic RNA ligand with an average length of ~400 base pairs. In the structure, the dimeric TLR3 units are clustered along the double-stranded RNA helix in a highly organized and cooperative fashion with a uniform inter-dimer spacing of 103 angstroms. The intracellular and transmembrane domains are dispensable for the clustering because their deletion does not interfere with the cluster formation. Our structural observation suggests that ligand-induced clustering of TLR3 dimers triggers the ordered assembly of intracellular signaling adaptors and initiates a robust innate immune response.
History
DepositionFeb 10, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateDec 7, 2022-
Current statusDec 7, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32851.png

Downloads & links

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Map

FileDownload / File: emd_32851.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCT-mut mutant (D523K,D524K,E527K) TLR3 ectodomain
Voxel sizeX=Y=Z: 0.8155 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.7439962 - 1.4538425
Average (Standard dev.)0.0005614049 (±0.029046364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 293.58002 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : CT-mut(D523K,D524K,E527K) TLR3 ectodomain-poly I:C complex

EntireName: CT-mut(D523K,D524K,E527K) TLR3 ectodomain-poly I:C complex
Components
  • Complex: CT-mut(D523K,D524K,E527K) TLR3 ectodomain-poly I:C complex
    • Complex: CT-mut(D523K,D524K,E527K) TLR3 ectodomain
      • Protein or peptide: Toll-like receptor 3
    • Complex: poly(I:C)
      • RNA: RNA (46-MER)
      • RNA: RNA (46-MER)

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Supramolecule #1: CT-mut(D523K,D524K,E527K) TLR3 ectodomain-poly I:C complex

SupramoleculeName: CT-mut(D523K,D524K,E527K) TLR3 ectodomain-poly I:C complex
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: CT-mut(D523K,D524K,E527K) TLR3 ectodomain

SupramoleculeName: CT-mut(D523K,D524K,E527K) TLR3 ectodomain / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: poly(I:C)

SupramoleculeName: poly(I:C) / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Macromolecule #1: Toll-like receptor 3

MacromoleculeName: Toll-like receptor 3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 77.539562 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: ADPKCTVSHE VADCSHLKLT QVPDDLPTNI TVLNLTHNQL RRLPAANFTR YSQLTSLDVG FNTISKLEPE LCQKLPMLKV LNLQHNELS QLSDKTFAFC TNLTELHLMS NSIQKIKNNP FVKQKNLITL DLSHNGLSST KLGTQVQLEN LQELLLSNNK I QALKSEEL ...String:
ADPKCTVSHE VADCSHLKLT QVPDDLPTNI TVLNLTHNQL RRLPAANFTR YSQLTSLDVG FNTISKLEPE LCQKLPMLKV LNLQHNELS QLSDKTFAFC TNLTELHLMS NSIQKIKNNP FVKQKNLITL DLSHNGLSST KLGTQVQLEN LQELLLSNNK I QALKSEEL DIFANSSLKK LELSSNQIKE FSPGCFHAIG RLFGLFLNNV QLGPSLTEKL CLELANTSIR NLSLSNSQLS TT SNTTFLG LKWTNLTMLD LSYNNLNVVG NDSFAWLPQL EYFFLEYNNI QHLFSHSLHG LFNVRYLNLK RSFTKQSISL ASL PKIDDF SFQWLKCLEH LNMEDNDIPG IKSNMFTGLI NLKYLSLSNS FTSLRTLTNE TFVSLAHSPL HILNLTKNKI SKIE SDAFS WLGHLEVLDL GLNEIGQELT GQEWRGLENI FEIYLSYNKY LQLTRNSFAL VPSLQRLMLR RVALKNVDSS PSPFQ PLRN LTILDLSNNN IANINKKMLK GLEKLEILDL QHNNLARLWK HANPGGPIYF LKGLSHLHIL NLESNGFDEI PVEVFK DLF ELKIIDLGLN NLNTLPASVF NNQVSLKSLN LQKNLITSVE KKVFGPAFRN LTELDMRFNP FDCTCESIAW FVNWINE TH TNIPELSSHY LCNTPPHYHG FPVRLFDTSS CKHHHHHH

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Macromolecule #2: RNA (46-MER)

MacromoleculeName: RNA (46-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.993408 KDa
SequenceString:
CCCCCCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCC

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Macromolecule #3: RNA (46-MER)

MacromoleculeName: RNA (46-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.143821 KDa
SequenceString:
IIIIIIIIII IIIIIIIIII IIIIIIIIII IIIIIIIIII IIIIII

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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

Concentration1.0 mg/mL
BufferpH: 5.5
Component:
ConcentrationFormulaName
20.0 mMC6H13NO4S2-(N-morpholino)ethanesulfonic acid
150.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 86284

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