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- PDB-7b1c: Cryo-EM of Aedes Aegypti Toll5A trimer bound to Spz1C -

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Basic information

Entry
Database: PDB / ID: 7b1c
TitleCryo-EM of Aedes Aegypti Toll5A trimer 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 / Cystine-knot cytokine / Leucine-rich repeat, SDS22-like subfamily / 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 / Cystine-knot cytokine / 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
TIR domain-containing protein / AAEL013433-PA
Similarity search - Component
Biological speciesAedes aegypti (yellow fever mosquito)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.74 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 / Category: citation / citation_author
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
C: Toll-like receptor
A: Toll-like receptor
D: AAEL013433-PA
P: AAEL013433-PA
B: Toll-like receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)296,17721
Polymers289,0245
Non-polymers7,15416
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 35 through 37 or (resid 38...
d_2ens_1(chain "B" and (resid 35 through 50 or (resid 51...
d_3ens_1(chain "C" and (resid 35 through 36 or (resid 37...
d_1ens_2(chain "E" and (resid 7 through 19 or (resid 20...
d_2ens_2(chain "F" and (resid 7 through 10 or (resid 11...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1CYSCYSA1 - 749
d_12ens_1NAGNAGB
d_13ens_1NAGNAGE
d_14ens_1NAGNAGG
d_15ens_1NAGNAGH
d_16ens_1NAGNAGI
d_21ens_1CYSCYSB2 - 750
d_22ens_1NAGNAGB
d_23ens_1NAGNAGB
d_24ens_1NAGNAGB
d_25ens_1NAGNAGB
d_26ens_1NAGNAGB
d_31ens_1CYSCYSC4 - 752
d_32ens_1NAGNAGC
d_33ens_1NAGNAGC
d_34ens_1NAGNAGC
d_35ens_1NAGNAGC
d_36ens_1NAGNAGC
d_11ens_2PROCYSE1 - 91
d_21ens_2PROCYSF1 - 91

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(0.700905564913, 0.381187711711, -0.602849332349), (0.375499131497, -0.915803125104, -0.142495748341), (-0.606408930771, -0.126493337732, -0.785027161435)92.8423457392, 262.366088565, 424.083743814
2given(-0.391394550273, -0.7148581847, 0.579472244188), (-0.832191565805, 0.00618965279666, -0.554453682467), (0.392769020951, -0.699242063928, -0.597321548427)225.072789917, 401.453673149, 302.064141363
3given(0.0655831029185, -0.614441728264, 0.786231657452), (-0.00740565556824, -0.78820599375, -0.61536693743), (0.997819629431, 0.0345351123381, -0.0562433386154)163.922398422, 413.738247023, -30.3462885163

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Components

#1: Protein Toll-like receptor /


Mass: 87675.773 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aedes aegypti (yellow fever mosquito) / Gene: 5569408 / Cell line (production host): S2 schneider cells / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: A0A6I8TEX2
#2: Protein AAEL013433-PA


Mass: 12998.096 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 line (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.: 6
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.: 7
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-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 / N-Acetylglucosamine


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
IDNameTypeDetailsEntity IDParent-IDSource
1Toll5A-Spz1C complexCOMPLEXHolodimer complex of Toll5A bound to Spz1C#1-#20MULTIPLE SOURCES
2Toll-like receptorCOMPLEX#11RECOMBINANT
3AAEL013433-PACOMPLEX#21RECOMBINANT
Molecular weightValue: 230 kDa/nm / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Aedes aegypti (yellow fever mosquito)7159
33Aedes aegypti (yellow fever mosquito)7159
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Drosophila melanogaster (fruit fly)7227
23Baculovirus expression vector pFastBac1-HM274590
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaClSodium chloride1
250 mMTris-HClTrisC4H11NO31
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 FIELDBright-field microscopy / Nominal defocus max: 2700 nm / Nominal defocus min: 1000 nm / Cs: 2 mm / C2 aperture diameter: 51.1 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_refine1.19rc4_4035refinement
PHENIX1.19rc4_4035refinement
EM software
IDNameCategory
2EPUimage acquisition
4WarpCTF correction
9cryoSPARCinitial Euler assignment
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 507268
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42866 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 191.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009517457
ELECTRON MICROSCOPYf_angle_d1.200923939
ELECTRON MICROSCOPYf_chiral_restr0.07433062
ELECTRON MICROSCOPYf_plane_restr0.00653138
ELECTRON MICROSCOPYf_dihedral_angle_d12.2675774
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints7.50767937915
ens_1d_3CELECTRON MICROSCOPYNCS constraints6.22674791592
ens_2d_2FELECTRON MICROSCOPYNCS constraints10.1847584518

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