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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)
KeywordsToll-like receptor / dsRNA / Innate immunity / Receptor / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex
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 / I-kappaB phosphorylation ...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 / I-kappaB phosphorylation / TLR3-mediated TICAM1-dependent programmed cell death / inflammatory response to wounding / toll-like receptor 3 signaling pathway / activation of NF-kappaB-inducing kinase activity / necroptotic signaling pathway / detection of virus / endolysosome membrane / RIP-mediated NFkB activation via ZBP1 / hyperosmotic response / positive regulation of cytokine production involved in inflammatory response / Trafficking and processing of endosomal TLR / positive regulation of macrophage cytokine production / cellular response to exogenous dsRNA / pattern recognition receptor activity / toll-like receptor signaling pathway / RSV-host interactions / 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 / JNK cascade / extrinsic apoptotic signaling pathway / TICAM1,TRAF6-dependent induction of TAK1 complex / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / positive regulation of interleukin-12 production / TICAM1-dependent activation of IRF3/IRF7 / positive regulation of interferon-beta production / extracellular matrix / TICAM1, RIP1-mediated IKK complex recruitment / positive regulation of interleukin-8 production / positive regulation of JNK cascade / microglial cell activation / cellular response to virus / cellular response to type II interferon / cellular response to mechanical stimulus / positive regulation of inflammatory response / positive regulation of interleukin-6 production / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of type II interferon production / positive regulation of angiogenesis / male gonad development / positive regulation of tumor necrosis factor production / double-stranded RNA binding / transmembrane signaling receptor activity / cellular response to xenobiotic stimulus / 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 / identical protein binding / membrane / 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 / TIR domain profile. / Leucine-rich repeat, SDS22-like subfamily ...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 / TIR domain profile. / Leucine-rich repeat, SDS22-like subfamily / 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-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 293.58 Å		0.82 Å/pix.
x 360 pix.
= 293.58 Å		0.82 Å/pix.
x 360 pix.
= 293.58 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8155 Å
Density

Histogram

Histogram (log scale)
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 / 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 / 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 / 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

UniProtKB: Toll-like receptor 3

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

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Image processing

Startup modelType of model: 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
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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