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- PDB-8y9b: TcdB1 in complex with mini-binder -

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Basic information

Entry
Database: PDB / ID: 8y9b
TitleTcdB1 in complex with mini-binder
Components
  • De novo design mini-binder
  • Toxin B
KeywordsTOXIN/DE NOVO PROTEIN / Mini-binder against C.difficle infection / TOXIN-DE NOVO PROTEIN complex
Function / homology
Function and homology information


glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis ...glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis / extracellular region / metal ion binding / membrane
Similarity search - Function
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. ...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. / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Biological speciesClostridioides difficile (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLv, X.C. / Lu, P.L.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0909200 China
National Natural Science Foundation of China (NSFC)32301219 China
CitationJournal: Nat Commun / Year: 2024
Title: De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants.
Authors: Xinchen Lv / Yuanyuan Zhang / Ke Sun / Qi Yang / Jianhua Luo / Liang Tao / Peilong Lu /
Abstract: Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal ...Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).
History
DepositionFeb 6, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Oct 16, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Toxin B
A: De novo design mini-binder
hetero molecules


Theoretical massNumber of molelcules
Total (without water)278,2703
Polymers278,2052
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Toxin B


Mass: 270767.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: tcdB, toxB / Production host: Bacillus subtilis (bacteria)
References: UniProt: P18177, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Protein De novo design mini-binder


Mass: 7437.460 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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

ComponentName: De novo design mini-binder in complex with TcdB1 / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Clostridioides difficile (bacteria)
Source (recombinant)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTrimethylol aminomethane hydrochlorideTris-HCl1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204441 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDChain-IDSource nameTypeAccession codeInitial refinement model-ID
1ARoseTTAFoldin silico model
27ml7

7ml7

BBPDBexperimental model7ml72
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413610
ELECTRON MICROSCOPYf_angle_d0.51918403
ELECTRON MICROSCOPYf_dihedral_angle_d4.3361781
ELECTRON MICROSCOPYf_chiral_restr0.0462086
ELECTRON MICROSCOPYf_plane_restr0.0032377

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