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- PDB-6wts: CryoEM structure of the C. sordellii lethal toxin TcsL in complex... -

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Basic information

Entry
Database: PDB / ID: 6wts
TitleCryoEM structure of the C. sordellii lethal toxin TcsL in complex with SEMA6A
Components
  • SEMA6A
  • TcsL
KeywordsSIGNALING PROTEIN/TOXIN / cryoEM / signaling protein-toxin complex
Function / homology
Function and homology information


negative regulation of cell adhesion involved in sprouting angiogenesis / semaphorin receptor binding / negative regulation of sprouting angiogenesis / Other semaphorin interactions / negative regulation of vascular endothelial growth factor signaling pathway / negative regulation of axon extension involved in axon guidance / positive regulation of neuron migration / chemorepellent activity / neural crest cell migration / host cell cytosol ...negative regulation of cell adhesion involved in sprouting angiogenesis / semaphorin receptor binding / negative regulation of sprouting angiogenesis / Other semaphorin interactions / negative regulation of vascular endothelial growth factor signaling pathway / negative regulation of axon extension involved in axon guidance / positive regulation of neuron migration / chemorepellent activity / neural crest cell migration / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / negative chemotaxis / glycosyltransferase activity / semaphorin-plexin signaling pathway / cellular response to vascular endothelial growth factor stimulus / cysteine-type peptidase activity / cytoskeleton organization / negative regulation of angiogenesis / host cell endosome membrane / axon guidance / animal organ morphogenesis / negative regulation of ERK1 and ERK2 cascade / nervous system development / toxin activity / cell surface receptor signaling pathway / positive regulation of cell migration / axon / apoptotic process / host cell plasma membrane / extracellular space / extracellular region / membrane / metal ion binding / plasma membrane
Similarity search - Function
Semaphorin / ligand-binding face of the semaphorins, domain 2 / ligand-binding face of the semaphorins, domain 2 / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain ...Semaphorin / ligand-binding face of the semaphorins, domain 2 / ligand-binding face of the semaphorins, domain 2 / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / Choline-binding repeat / Putative cell wall binding repeat / Cell wall/choline-binding repeat / Cell wall-binding repeat profile. / Plexin repeat / Plexin repeat / Sema domain / semaphorin domain / Sema domain / Sema domain superfamily / Sema domain profile. / PSI domain / domain found in Plexins, Semaphorins and Integrins / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / Nucleotide-diphospho-sugar transferases / WD40/YVTN repeat-like-containing domain superfamily / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Semaphorin-6A / Lethal Toxin
Similarity search - Component
Biological speciesHomo sapiens (human)
Paeniclostridium sordellii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKucharska, I. / Rubinstein, J.L. / Julien, J.P.
Funding support Canada, 3items
OrganizationGrant numberCountry
Other governmentOntario Early Researcher Awards program Canada
Other governmentCanada Foundation for Innovation Canada
Other governmentOntario Research Fund Canada
CitationJournal: Cell / Year: 2020
Title: Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.
Authors: Hunsang Lee / Greg L Beilhartz / Iga Kucharska / Swetha Raman / Hong Cui / Mandy Hiu Yi Lam / Huazhu Liang / John L Rubinstein / Daniel Schramek / Jean-Philippe Julien / Roman A Melnyk / Mikko Taipale /
Abstract: Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome ...Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
History
DepositionMay 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_entity_id / _chem_comp.name / _pdbx_entity_nonpoly.entity_id / _pdbx_entity_nonpoly.name / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Aug 5, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: SEMA6A
B: SEMA6A
C: TcsL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,18910
Polymers191,0313
Non-polymers2,1587
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6050 Å2
ΔGint6 kcal/mol
Surface area57710 Å2

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Components

#1: Protein SEMA6A / Semaphorin VIA / Sema VIA / Semaphorin-6A-1 / SEMA6A-1 / Semaphorin-6A


Mass: 63857.410 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEMA6A, KIAA1368, SEMAQ / Production host: Homo sapiens (human) / References: UniProt: Q9H2E6
#2: Protein TcsL / Lethal Toxin / Cytotoxin L


Mass: 63315.875 Da / Num. of mol.: 1 / Fragment: UNP residues 1285-1804
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paeniclostridium sordellii (bacteria) / Gene: tcsL, JGS6382_PCS1300041 / Production host: Escherichia coli (E. coli)
References: UniProt: V5T923, Transferases; Glycosyltransferases; Hexosyltransferases
#3: 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.: 3
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
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
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
1Complex of SEMA6A with TcsLCOMPLEX#1-#20MULTIPLE SOURCES
2SEMA6ACOMPLEX#11RECOMBINANT
3TcsLCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Paeniclostridium sordellii (bacteria)1505
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 45.24 e/Å2 / Film or detector model: OTHER / Details: Dataset was collected with FEI Falcon IV.

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Processing

SoftwareName: PHENIX / Version: 1.18rc5_3822: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2image acquisition
4cryoSPARC2CTF correction
7UCSF Chimera1.14model fitting
11cryoSPARC2classification
13PHENIX1.18model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179188 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6C0B
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 76.11 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210084
ELECTRON MICROSCOPYf_angle_d0.46213695
ELECTRON MICROSCOPYf_dihedral_angle_d15.0333531
ELECTRON MICROSCOPYf_chiral_restr0.0431575
ELECTRON MICROSCOPYf_plane_restr0.0041740

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