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- PDB-7b1b: Cryo-EM of Aedes Aegypti Toll5A dimer bound to Spz1C -

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Basic information

Entry
Database: PDB / ID: 7b1b
TitleCryo-EM of Aedes Aegypti Toll5A dimer bound to Spz1C
Components
  • AAEL013433-PA
  • Toll-like receptor
KeywordsIMMUNE SYSTEM / Mosquito vector biology / Toll receptor / LRR / Cystine knot domain
Function / homology
Function and homology information


toll-like receptor signaling pathway / transmembrane signaling receptor activity / immune response / extracellular space / membrane
Similarity search - Function
Spaetzle / Spaetzle / Toll-like receptor / TIR domain / Leucine-rich repeats, bacterial type / Toll - interleukin 1 - resistance / Leucine-rich repeat, SDS22-like subfamily / Cystine-knot cytokine / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain ...Spaetzle / Spaetzle / Toll-like receptor / TIR domain / Leucine-rich repeats, bacterial type / Toll - interleukin 1 - resistance / Leucine-rich repeat, SDS22-like subfamily / Cystine-knot cytokine / 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
TIR domain-containing protein / AAEL013433-PA
Similarity search - Component
Biological speciesAedes aegypti (yellow fever mosquito)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.23 Å
AuthorsGangloff, M. / Hardwick, S.W. / Chirgadze, D.Y.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P02260X/1 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Structure and dynamics of Toll immunoreceptor activation in the mosquito Aedes aegypti.
Authors: Yoann Saucereau / Thomas H Wilson / Matthew C K Tang / Martin C Moncrieffe / Steven W Hardwick / Dimitri Y Chirgadze / Sandro G Soares / Maria Jose Marcaida / Nicholas J Gay / Monique Gangloff /
Abstract: Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone ...Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone duplication which may allow neofunctionalization and adaptation. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A complexes that display transient but specific interactions with Spaetzle1C, forming asymmetric complexes, with only one ligand clearly resolved. Loop structures of Spaetzle1C and Toll5A intercalate, temporarily bridging the receptor C-termini to promote signalling. By contrast unbound receptors form head-to-head homodimers that keep the juxtamembrane regions far apart in an inactive conformation. Interestingly the transcriptional signature of Spaetzle1C differs from other Spaetzle cytokines and controls genes involved in innate immunity, metabolism and tissue regeneration. Taken together our results explain how upregulation of Spaetzle1C in the midgut and Toll5A in the salivary gland shape the concomitant immune response.
History
DepositionNov 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toll-like receptor
D: AAEL013433-PA
P: AAEL013433-PA
B: Toll-like receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,67914
Polymers201,2344
Non-polymers4,44510
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, gel filtration, mass spectrometry, surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "B" and (resid 32 through 52 or (resid 53...
d_2ens_1(chain "C" and (resid 32 through 37 or (resid 38...
d_1ens_2(chain "E" and (resid 6 through 19 or (resid 20...
d_2ens_2(chain "F" and (resid 6 through 8 or (resid 9...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ARGCYSB1 - 752
d_12ens_1NAGNAGB
d_13ens_1NAGNAGB
d_14ens_1NAGNAGB
d_15ens_1NAGNAGB
d_16ens_1NAGNAGB
d_17ens_1NAGNAGB
d_18ens_1BMABMAB
d_21ens_1ARGCYSC2 - 753
d_22ens_1NAGNAGC
d_23ens_1NAGNAGC
d_24ens_1NAGNAGC
d_25ens_1NAGNAGC
d_26ens_1NAGNAGC
d_27ens_1NAGNAGC
d_28ens_1BMABMAC
d_11ens_2ALACYSE1 - 92
d_21ens_2ALACYSF1 - 92

NCS ensembles :
ID
ens_1
ens_2

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Components

#1: Protein Toll-like receptor


Mass: 87675.773 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aedes aegypti (yellow fever mosquito) / Gene: 5569408 / Plasmid: pMT-V5-HisA / Cell line (production host): S2 / Production host: Drosophila melanocephala (fry) / References: UniProt: A0A6I8TEX2
#2: Protein AAEL013433-PA


Mass: 12941.044 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aedes aegypti (yellow fever mosquito) / Gene: SPZ1C, AAEL013433 / Plasmid: pFastBac-1 / Cell (production host): Sf9 / Production host: Baculovirus expression vector pFastBac1-HM / References: UniProt: Q16J57
#3: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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
1Cryo-EM of Aedes Aegypti Toll5A dimer bound to Spz1CCOMPLEX#1-#20RECOMBINANT
2Toll-like receptorCOMPLEX#11RECOMBINANT
3AAEL013433-PACOMPLEX#21RECOMBINANT
Molecular weightValue: 0.230 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Aedes aegypti (yellow fever mosquito)7159
23Aedes aegypti (yellow fever mosquito)7159
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Drosophila melanocephala (fry)46804
23Baculovirus expression vector pFastBac1-HM274590
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
250 mMTris-HClC4H11NO31
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K / Details: Blotting force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 51.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20rc3_4406refinement
PHENIX1.20rc3_4406refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4WarpCTF correction
7Coot0.9model fitting
9cryoSPARCinitial Euler assignment
12cryoSPARC3D reconstruction
13PHENIX1.20rc3_4406model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 507268
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40153 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
14LXR

4lxr

A1
24LXR

4lxr

J1
34LXR

4lxr

K1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 243.23 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003913099
ELECTRON MICROSCOPYf_angle_d0.657117886
ELECTRON MICROSCOPYf_chiral_restr0.04272226
ELECTRON MICROSCOPYf_plane_restr0.00382302
ELECTRON MICROSCOPYf_dihedral_angle_d6.54961925
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints8.31793684698
ens_2d_2CELECTRON MICROSCOPYNCS constraints9.39972765577

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