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- EMDB-37794: Cryo-EM Structure of Mouse TLR4/MD-2/DLAM3 Complex -

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Entry
Database: EMDB / ID: EMD-37794
TitleCryo-EM Structure of Mouse TLR4/MD-2/DLAM3 Complex
Map datamain map
Sample
  • Complex: mouse TLR4/MD2/DLAM3 complex
    • Protein or peptide: Toll-like receptor 4
    • Protein or peptide: Lymphocyte antigen 96
  • Ligand: (3R)-3-(dodecanoyloxy)tetradecanoic acid
  • Ligand: (3R)-3-(tetradecanoyloxy)tetradecanoic acid
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose
  • Ligand: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one
KeywordsInnate immune system / Toll-like receptors / TLR4 agonist / Vaccine adjuvants / Disaccharide-based Lipid A Mimetics / IMMUNE SYSTEM
Function / homology
Function and homology information


MyD88-independent TLR4 cascade / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade / Heme signaling / nitric oxide production involved in inflammatory response / Regulation of TLR by endogenous ligand / MHC class II biosynthetic process / TRIF-mediated programmed cell death / positive regulation of cellular response to macrophage colony-stimulating factor stimulus / TRAF6-mediated induction of TAK1 complex within TLR4 complex ...MyD88-independent TLR4 cascade / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade / Heme signaling / nitric oxide production involved in inflammatory response / Regulation of TLR by endogenous ligand / MHC class II biosynthetic process / TRIF-mediated programmed cell death / positive regulation of cellular response to macrophage colony-stimulating factor stimulus / TRAF6-mediated induction of TAK1 complex within TLR4 complex / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / lipopolysaccharide immune receptor activity / positive regulation of nucleotide-binding oligomerization domain containing 1 signaling pathway / positive regulation of matrix metallopeptidase secretion / IKK complex recruitment mediated by RIP1 / Toll-like receptor 4 binding / mast cell activation / detection of lipopolysaccharide / positive regulation of lymphocyte proliferation / regulation of dendritic cell cytokine production / lipopolysaccharide receptor complex / negative regulation of interleukin-23 production / cellular response to oxidised low-density lipoprotein particle stimulus / wound healing involved in inflammatory response / B cell proliferation involved in immune response / lymphocyte proliferation / nucleotide-binding oligomerization domain containing 1 signaling pathway / positive regulation of nucleotide-binding oligomerization domain containing 2 signaling pathway / positive regulation of stress-activated MAPK cascade / activation of NF-kappaB-inducing kinase activity / intestinal epithelial structure maintenance / positive regulation of interleukin-13 production / positive regulation of interleukin-1 production / TRIF-dependent toll-like receptor signaling pathway / macrophage activation / astrocyte development / microglia differentiation / nucleotide-binding oligomerization domain containing 2 signaling pathway / positive regulation of MHC class II biosynthetic process / NAD+ nucleosidase activity, cyclic ADP-ribose generating / positive regulation of platelet activation / positive regulation of macrophage activation / negative regulation of interleukin-17 production / positive regulation of lipopolysaccharide-mediated signaling pathway / positive regulation of cytokine production involved in inflammatory response / positive regulation of chemokine (C-X-C motif) ligand 2 production / negative regulation of cold-induced thermogenesis / positive regulation of extrinsic apoptotic signaling pathway / positive regulation of smooth muscle cell migration / MyD88-dependent toll-like receptor signaling pathway / toll-like receptor 4 signaling pathway / toll-like receptor signaling pathway / positive regulation of macrophage cytokine production / positive regulation of NLRP3 inflammasome complex assembly / cellular response to lipoteichoic acid / positive regulation of reactive oxygen species biosynthetic process / negative regulation of type II interferon production / negative regulation of interleukin-6 production / B cell proliferation / positive regulation of interferon-alpha production / positive regulation of interleukin-10 production / negative regulation of tumor necrosis factor production / phagocytosis / stress-activated MAPK cascade / phagocytic cup / positive regulation of chemokine production / cellular response to platelet-derived growth factor stimulus / ruffle / positive regulation of B cell proliferation / JNK cascade / ERK1 and ERK2 cascade / neurogenesis / nitric oxide biosynthetic process / positive regulation of interleukin-12 production / positive regulation of smooth muscle cell proliferation / activation of innate immune response / positive regulation of interferon-beta production / lipopolysaccharide-mediated signaling pathway / positive regulation of interleukin-1 beta production / positive regulation of interleukin-8 production / positive regulation of JNK cascade / response to bacterium / lipopolysaccharide binding / cellular response to mechanical stimulus / positive regulation of non-canonical NF-kappaB signal transduction / : / negative regulation of ERK1 and ERK2 cascade / positive regulation of interleukin-6 production / cellular response to type II interferon / positive regulation of type II interferon production / cellular response to amyloid-beta / positive regulation of nitric oxide biosynthetic process / positive regulation of inflammatory response / positive regulation of tumor necrosis factor production / transmembrane signaling receptor activity / cellular response to lipopolysaccharide / regulation of inflammatory response / response to lipopolysaccharide
Similarity search - Function
Lymphocyte antigen 96 / MD-2-related lipid-recognition domain / Domain involved in innate immunity and lipid metabolism. / Toll-like receptor / TIR domain / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain ...Lymphocyte antigen 96 / MD-2-related lipid-recognition domain / Domain involved in innate immunity and lipid metabolism. / Toll-like receptor / TIR domain / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance / 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 / Leucine-rich repeat domain superfamily / Immunoglobulin E-set
Similarity search - Domain/homology
Lymphocyte antigen 96 / Toll-like receptor 4
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsFu Y / Kim H / Kim HM
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
Other governmentInstitute for Basic Science, IBS Korea, Republic Of
CitationJournal: Nat Commun / Year: 2025
Title: Structural insight into TLR4/MD-2 activation by synthetic LPS mimetics with distinct binding modes.
Authors: Yaoyao Fu / Hyojin Kim / Dong Sun Lee / Ah-Reum Han / Holger Heine / Alla Zamyatina / Ho Min Kim /
Abstract: The mammalian pattern-recognition receptor TLR4/MD-2 (Toll-like receptor 4/myeloid differentiation factor-2) can be activated by a wide variety of pathogen-associated and endogenous molecules, with ...The mammalian pattern-recognition receptor TLR4/MD-2 (Toll-like receptor 4/myeloid differentiation factor-2) can be activated by a wide variety of pathogen-associated and endogenous molecules, with Gram-negative bacterial lipopolysaccharide (LPS) being the primary natural TLR4 agonist. Activation of TLR4 triggers cellular signaling that enables the beneficial innate immune responses and enhances adaptive immunity, thereby emphasizing the potential of TLR4 agonists for the management of diseases with an immunopathological background and for use as vaccine adjuvants. Given the challenges associated with LPS-derived products, including structural complexity, heterogeneity, toxicity, and species specificity, synthetic molecules targeting TLR4/MD-2 offer a promising alternative. Here, we elucidate the structural basis for the recognition of synthetic LPS-mimicking glycolipids, Disaccharide Lipid A Mimetics (DLAMs), by human and mouse TLR4/MD-2 through cryo-EM structures of six dimeric [TLR4/MD-2/ligand] complexes resolved at 2.2-3.1 Å. We reveal that the specific binding modes of DLAMs, distinct from those of LPS, are essential for the species-independent TLR4 agonistic activity. DLAMs function as a molecular bridge, effectively induce the dimerization of TLR4/MD-2 complexes through specific carbohydrate structure-relevant ligand-protein interactions. Our findings reveal the distinct molecular modes of TLR4 activation, and provide a structural basis for the rationale design and development of innovative, highly potent TLR4-targeting immunotherapeutics and adjuvants.
History
DepositionOct 16, 2023-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37794.png

Downloads & links

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Map

FileDownload / File: emd_37794.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 400 pix.
= 339.2 Å		0.85 Å/pix.
x 400 pix.
= 339.2 Å		0.85 Å/pix.
x 400 pix.
= 339.2 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.848 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.75979394 - 1.939884
Average (Standard dev.)-0.0014122202 (±0.03537253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 339.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half B map

Fileemd_37794_half_map_1.map
Annotationhalf B map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half A map

Fileemd_37794_half_map_2.map
Annotationhalf A map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : mouse TLR4/MD2/DLAM3 complex

EntireName: mouse TLR4/MD2/DLAM3 complex
Components
  • Complex: mouse TLR4/MD2/DLAM3 complex
    • Protein or peptide: Toll-like receptor 4
    • Protein or peptide: Lymphocyte antigen 96
  • Ligand: (3R)-3-(dodecanoyloxy)tetradecanoic acid
  • Ligand: (3R)-3-(tetradecanoyloxy)tetradecanoic acid
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose
  • Ligand: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one

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Supramolecule #1: mouse TLR4/MD2/DLAM3 complex

SupramoleculeName: mouse TLR4/MD2/DLAM3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Lymphocyte antigen 96

MacromoleculeName: Lymphocyte antigen 96 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 16.411711 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
EKQQWFCNSS DAIISYSYCD HLKFPISISS EPCIRLRGTN GFVHVEFIPR GNLKYLYFNL FISVNSIELP KRKEVLCHGH DDDYSFCRA LKGETVNTSI PFSFEGILFP KGHYRCVAEA IAGDTEEKLF CLNFTIIHRR DVN

UniProtKB: Lymphocyte antigen 96

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Macromolecule #2: Toll-like receptor 4

MacromoleculeName: Toll-like receptor 4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 68.651281 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: NPCIEVVPNI TYQCMDQKLS KVPDDIPSST KNIDLSFNPL KILKSYSFSN FSELQWLDLS RCEIETIEDK AWHGLHHLSN LILTGNPIQ SFSPGSFSGL TSLENLVAVE TKLASLESFP IGQLITLKKL NVAHNFIHSC KLPAYFSNLT NLVHVDLSYN Y IQTITVND ...String:
NPCIEVVPNI TYQCMDQKLS KVPDDIPSST KNIDLSFNPL KILKSYSFSN FSELQWLDLS RCEIETIEDK AWHGLHHLSN LILTGNPIQ SFSPGSFSGL TSLENLVAVE TKLASLESFP IGQLITLKKL NVAHNFIHSC KLPAYFSNLT NLVHVDLSYN Y IQTITVND LQFLRENPQV NLSLDMSLNP IDFIQDQAFQ GIKLHELTLR GNFNSSNIMK TCLQNLAGLH VHRLILGEFK DE RNLEIFE PSIMEGLCDV TIDEFRLTYT NDFSDDIVKF HCLANVSAMS LAGVSIKYLE DVPKHFKWQS LSIIRCQLKQ FPT LDLPFL KSLTLTMNKG SISFKKVALP SLSYLDLSRN ALSFSGCCSY SDLGTNSLRH LDLSFNGAII MSANFMGLEE LQHL DFQHS TLKRVTEFSA FLSLEKLLYL DISYTNTKID FDGIFLGLTS LNTLKMAGNS FKDNTLSNVF ANTTNLTFLD LSKCQ LEQI SWGVFDTLHR LQLLNMSHNN LLFLDSSHYN QLYSLSTLDC SFNRIETSKG ILQHFPKSLA FFNLTNNSVA CICEHQ KFL QWVKEQKQFL VNVEQMTCAT PVEMNTSLVL DFNNSTCYMY K

UniProtKB: Toll-like receptor 4

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Macromolecule #4: (3R)-3-(dodecanoyloxy)tetradecanoic acid

MacromoleculeName: (3R)-3-(dodecanoyloxy)tetradecanoic acid / type: ligand / ID: 4 / Number of copies: 4 / Formula: 2IL
Molecular weightTheoretical: 426.673 Da
Chemical component information

ChemComp-2IL:
(3R)-3-(dodecanoyloxy)tetradecanoic acid

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Macromolecule #5: (3R)-3-(tetradecanoyloxy)tetradecanoic acid

MacromoleculeName: (3R)-3-(tetradecanoyloxy)tetradecanoic acid / type: ligand / ID: 5 / Number of copies: 2 / Formula: 0IL
Molecular weightTheoretical: 454.726 Da

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 16 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #7: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose

MacromoleculeName: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 2 / Formula: GP4
Molecular weightTheoretical: 259.151 Da
Chemical component information

ChemComp-GP4:
2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose

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Macromolecule #8: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one

MacromoleculeName: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one
type: ligand / ID: 8 / Number of copies: 2 / Formula: XIQ
Molecular weightTheoretical: 188.135 Da

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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

BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
200.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec. / Details: 10mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 18733 / Average electron dose: 66.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 58963 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3vq2

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 625831
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3vq2


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-8wry:
Cryo-EM Structure of Mouse TLR4/MD-2/DLAM3 Complex

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