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- PDB-9dlg: CryoEM structure of the TIR domain from human TRAM -

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Basic information

Entry
Database: PDB / ID: 9dlg
TitleCryoEM structure of the TIR domain from human TRAM
ComponentsTIR domain-containing adapter molecule 2
KeywordsIMMUNE SYSTEM / Toll-like receptor / TIR / TRAM
Function / homology
Function and homology information


TRAM-dependent toll-like receptor 4 signaling pathway / positive regulation of interleukin-18-mediated signaling pathway / negative regulation of chemokine (C-C motif) ligand 5 production / MyD88-independent TLR4 cascade / negative regulation of toll-like receptor 4 signaling pathway / positive regulation of toll-like receptor 4 signaling pathway / TRIF-mediated programmed cell death / positive regulation of chemokine (C-C motif) ligand 5 production / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade ...TRAM-dependent toll-like receptor 4 signaling pathway / positive regulation of interleukin-18-mediated signaling pathway / negative regulation of chemokine (C-C motif) ligand 5 production / MyD88-independent TLR4 cascade / negative regulation of toll-like receptor 4 signaling pathway / positive regulation of toll-like receptor 4 signaling pathway / TRIF-mediated programmed cell death / positive regulation of chemokine (C-C motif) ligand 5 production / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade / TRIF-dependent toll-like receptor signaling pathway / toll-like receptor 4 signaling pathway / phagocytic cup / positive regulation of type I interferon production / phagocytosis / signaling adaptor activity / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / TRAF6-mediated induction of TAK1 complex within TLR4 complex / secretory granule membrane / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / IKK complex recruitment mediated by RIP1 / cell projection / late endosome membrane / late endosome / cellular response to lipopolysaccharide / early endosome membrane / molecular adaptor activity / positive regulation of canonical NF-kappaB signal transduction / early endosome / endosome / endosome membrane / inflammatory response / innate immune response / Neutrophil degranulation / endoplasmic reticulum / Golgi apparatus / plasma membrane
Similarity search - Function
: / TIR domain / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily
Similarity search - Domain/homology
TIR domain-containing adapter molecule 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsHedger, A. / Pospich, S. / Pan, M. / Gu, W. / Ve, T. / Raunser, S. / Landsberg, M. / Nanson, J.D. / Kobe, B.
Funding support Australia, 5items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)2025931 Australia
National Health and Medical Research Council (NHMRC, Australia)1196590 Australia
Australian Research Council (ARC)FL180100109 Australia
Australian Research Council (ARC)FT200100572 Australia
Australian Research Council (ARC)DE170100783 Australia
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for TIR domain-mediated innate immune signaling by Toll-like receptor adaptors TRIF and TRAM.
Authors: Mohammad K Manik / Mengqi Pan / Le Xiao / Weixi Gu / Hyoyoung Kim / Sabrina Pospich / Andrew Hedger / Parimala R Vajjhala / Morris Y L Lee / Xiaoqi Qian / Michael J Landsberg / Thomas Ve / ...Authors: Mohammad K Manik / Mengqi Pan / Le Xiao / Weixi Gu / Hyoyoung Kim / Sabrina Pospich / Andrew Hedger / Parimala R Vajjhala / Morris Y L Lee / Xiaoqi Qian / Michael J Landsberg / Thomas Ve / Jeffrey D Nanson / Stefan Raunser / Katryn J Stacey / Hao Wu / Bostjan Kobe /
Abstract: Innate immunity relies on Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns. The TIR (Toll/interleukin-1 receptor) domain-containing TLR adaptors TRIF (TIR domain-containing ...Innate immunity relies on Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns. The TIR (Toll/interleukin-1 receptor) domain-containing TLR adaptors TRIF (TIR domain-containing adaptor-inducing interferon-β) and TRAM (TRIF-related adaptor molecule) are essential for MyD88-independent TLR signaling. However, the structural basis of TRIF and TRAM TIR domain-based signaling remains unclear. Here, we present cryo-EM structures of filaments formed by TRIF and TRAM TIR domains at resolutions of 3.3 Å and 5.6 Å, respectively. Both structures reveal two-stranded parallel helical arrangements. Functional studies underscore the importance of intrastrand interactions, mediated by the BB-loop, and interstrand interactions in TLR4-mediated signaling. We also report the crystal structure of the monomeric TRAM TIR domain bearing the BB loop mutation C117H, which reveals conformational differences consistent with its inactivity. Our findings suggest a unified signaling mechanism by the TIR domains of the four signaling TLR adaptors MyD88, MAL, TRIF, and TRAM and reveal potential therapeutic targets for immunity-related disorders.
History
DepositionSep 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2025Group: Data collection / Data processing / Category: em_3d_reconstruction / em_admin
Item: _em_3d_reconstruction.num_particles / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: TIR domain-containing adapter molecule 2
A: TIR domain-containing adapter molecule 2
C: TIR domain-containing adapter molecule 2
D: TIR domain-containing adapter molecule 2
E: TIR domain-containing adapter molecule 2


Theoretical massNumber of molelcules
Total (without water)91,7545
Polymers91,7545
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "A"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: VAL / Beg label comp-ID: VAL / End auth comp-ID: ARG / End label comp-ID: ARG / Auth seq-ID: 77 - 231 / Label seq-ID: 2 - 156

Dom-IDAuth asym-IDLabel asym-ID
d_1BA
d_2AB
d_3CC
d_4DD
d_5EE

NCS oper:
IDCodeMatrixVector
1given(0.998024055293, 0.0600040799796, -0.0186412296313), (-0.0594834291516, 0.997855070414, 0.0273309367777), (0.0202412132227, -0.0261680880958, 0.99945261241)-4.09825329395, 4.35137774483, 34.2519445423
2given(0.998934404955, -0.046090431013, 0.00239306627379), (0.0460494218084, 0.998826060308, 0.0150316998281), (-0.00308307448224, -0.014905482805, 0.999884153907)5.46163482254, -8.42217416129, -30.7263303371
3given(-0.997454084363, -0.00726820051236, 0.0709402766385), (0.00270220843998, -0.99793025234, -0.0642488095913), (0.0712604213977, -0.063893542128, 0.995409246299)276.077176361, 290.728751356, 17.0178296075
4given(-0.996421823388, 0.0595985695243, 0.0599296286299), (-0.0635226972653, -0.995804992759, -0.0658580543918), (0.0557531775705, -0.0694292942985, 0.996027688513)266.097538225, 301.267375533, -13.3919436706

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Components

#1: Protein
TIR domain-containing adapter molecule 2 / TICAM-2 / Putative NF-kappa-B-activating protein 502 / TRIF-related adapter molecule / TRAM / Toll- ...TICAM-2 / Putative NF-kappa-B-activating protein 502 / TRIF-related adapter molecule / TRAM / Toll-like receptor adaptor protein 3 / Toll/interleukin-1 receptor domain-containing protein / MyD88-4


Mass: 18350.752 Da / Num. of mol.: 5 / Fragment: TIR domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TICAM2, TIRAP3, TIRP, TRAM / Production host: Escherichia coli (E. coli) / References: UniProt: Q86XR7
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Self-assembly of TIR domain of TRIF-related adaptor protein (TRAM)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 50000 nm / Nominal defocus min: 3000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 177.9 ° / Axial rise/subunit: 16.11 Å / Axial symmetry: C1
3D reconstructionResolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95000 / Symmetry type: HELICAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 283.85 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00176560
ELECTRON MICROSCOPYf_angle_d0.45348905
ELECTRON MICROSCOPYf_chiral_restr0.0378975
ELECTRON MICROSCOPYf_plane_restr0.00651180
ELECTRON MICROSCOPYf_dihedral_angle_d13.9362470
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2ABELECTRON MICROSCOPYNCS constraints1.4832305722E-11
ens_1d_3ABELECTRON MICROSCOPYNCS constraints1.29955530567E-12
ens_1d_4ABELECTRON MICROSCOPYNCS constraints6.8221320358E-12
ens_1d_5ABELECTRON MICROSCOPYNCS constraints4.31847214136E-12

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