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- EMDB-39073: De novo design mini-binder in complex with TcdB4 -

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Basic information

Entry
Database: EMDB / ID: EMD-39073
TitleDe novo design mini-binder in complex with TcdB4
Map data
Sample
  • Complex: TcdB4 in complex with De novo design mini-binder
    • Protein or peptide: Toxin B
    • Protein or peptide: De novo design Minibinder
  • Ligand: ZINC ION
KeywordsDe novo design mini-binder against C.difficle toxin B / DE NOVO PROTEIN / TOXIN-DE NOVO PROTEIN complex
Biological speciesClostridioides difficile (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLv XC / Lu PL
Funding support China, 2 items
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, 2024-
Header (metadata) releaseAug 28, 2024-
Map releaseAug 28, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39073.png

Downloads & links

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Map

FileDownload / File: emd_39073.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 344.736 Å		1.08 Å/pix.
x 320 pix.
= 344.736 Å		1.08 Å/pix.
x 320 pix.
= 344.736 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0773 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-3.8220153 - 6.058354
Average (Standard dev.)-0.0006746466 (±0.11663893)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 344.736 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : TcdB4 in complex with De novo design mini-binder

EntireName: TcdB4 in complex with De novo design mini-binder
Components
  • Complex: TcdB4 in complex with De novo design mini-binder
    • Protein or peptide: Toxin B
    • Protein or peptide: De novo design Minibinder
  • Ligand: ZINC ION

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Supramolecule #1: TcdB4 in complex with De novo design mini-binder

SupramoleculeName: TcdB4 in complex with De novo design mini-binder / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Clostridioides difficile (bacteria)

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Macromolecule #1: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 1 / Details: Genbank Sequence ID MCP3315766.1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 270.454 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MSLVNRKQLE KMANVRFRVQ EDEYVAILDA LEEYHNMSEN TVVEKYLKLK DINSLTDTYI DTYKKSGRNK ALKKFKEYLV TEILELKNS NLTPVEKNLH FIWIGGQIND TAINYINQWK DVNSDYNVNV FYDSNAFLIN TLKKTIIESA SNDTLESFRE N LNDPEFNH ...String:
MSLVNRKQLE KMANVRFRVQ EDEYVAILDA LEEYHNMSEN TVVEKYLKLK DINSLTDTYI DTYKKSGRNK ALKKFKEYLV TEILELKNS NLTPVEKNLH FIWIGGQIND TAINYINQWK DVNSDYNVNV FYDSNAFLIN TLKKTIIESA SNDTLESFRE N LNDPEFNH TAFFRKRMQI IYDKQQNFIN YYKAQKEENP DLIIDDIVKT YLSNEYSKDI DELNAYIEES LNKVTENSGN DV RNFEEFK TGEVFNLYEQ ELVERWNLAG ASDILRVAIL KNIGGVYLDV DMLPGIHPDL FKDINKPDSV KTAVDWEEMQ LEA IMKYKE YIPEYTSKHF DTLDEEVQSS FESVLASKSD KSEIFLPLGD IEVSPLEVKV AFAKGSIINQ ALISAKDSYC SDLL IKQIQ NRYKILNDTL GPIISQGNDF NTTMNNFGES LGAIANEENI SFIAKIGSYL RVGFYPEANT TITLSGPTIY AGAYK DLLT FKEMSIDTSI LSSELRNFEF PKVNISQATE QEKNSLWQFN EERAKIQFEE YKKNYFEGAL GEDDNLDFSQ NTVTDK EYL LEKISSSTKS SERGYVHYIV QLQGDKISYE AACNLFAKNP YDSILFQKNI EDSEVAYYYN PTDSEIQEID KYRIPDR IS DRPKIKLTLI GHGKAEFNTD IFAGLDVDSL SSEIETIIDL AKADISPKSI EINLLGCNMF SYSVNVEETY PGKLLLRV K DKVSELMPSI SQDSIIVSAN QYEVRINSEG RRELLDHSGE WINKEESIIK DISSKEYISF NPKENKIIVK SKNLPELST LLQEIRNNSN SSDIELEEKV MLAECEINVI SNIETQVVEE RIEEAKSLTS DSINYIKNEF KLIESISDAL YDLKQQNELE ESHFISFED ISKTDEGFSI RFIDKETGES IFVETEKAIF SEYANHITEE ISKLKDTIFD TVNGKLVKKV TLDATHEVNT L NAAFFIQS LIGYNSSKES LSNLSVAMKV QVYAQLFSTG LNTITDAAKV VELVSTALDE TIDLLPTLSE GLPVIATIID GV SLGASIK ELSETSDPLL RQEIEAKIGI MAVNLTAATT AIITSSLGIA SGFSILLVPL AGISAGIPSL VNNELILRAE AKN VVDYFG HISLAESEGA FTLLDDKIMM PQDDLVISEI DFNNNSITLG KCEIWRMEGG SGHTVTDDID HFFSAPSTTY REPY LSIYD VLDVKKEELD LSKDLMVLPN APDRIFGWER GWTPGLRSLE NDGTKLLDRI RDHYEGQFYW RFFAFIADSV ITKLK PRYE DTNIRISLDS NTRSFIVPVI TTEYIREKLS YSFYGSGGTY ALSLSQYNMN INIELNENDT WVIDVDNVVR DVTIES DKI KKGDLIENIL SKLSIEDNKI ILDNHEINFS GTLNGGNGFV SLTFSILEGI NAVIEVDLLS KSYKVLISGE LKTLMAN SN SVQQKIDYIG LNSELQKNIP YSFMDDEGKE NGFINCFTKE GLFVSELSDV VLIIKVYMDN SKPPFGYYSN DLKDVKVI T KDDVIILTGY YLKDDIKISL SFTIQDKNTI KLNGVYLDEN GVAEILKFMN KKGSTNTSDS LMSFLESMNI KSIFIKSLK SNAKLILDTN FIISGTTSIG QFEFICDKDN NIQPYFIKFN TLETKYTLYV GNRQNMIVEP NYNLDDSGDI SSTVINFSQK YLYGIDSCV NKVIISPNIY TDEINITPVH EANNTYPEVI VLDTNYISEK INININDLSI RYVWSNDGSD FILMSTDEEN K VSQVKIRF TNVFKGNTIS DKISFNFSDK QDISINKIIS TFTPSYYVEG LLNYDLGLIS LYNEKFYINN LGMMVSGLVY IN DSLYYFK PPIKNLITGF TTIGDDKYYF NPDNGGAASV GETIIDGKNY YFSQNGVLQT GVFSTEDGFK YFAPADTLDE NLE GEAIDF TGKLIIDENV YYFGDNYRAA IEWQTLDDEV YYFSTDTGRA FKGLNQIGDD KFYFNSDGIM QKGFVNINDK TFYF DDSGV MKSGYTEIDG KYFYFAENGE MQIGVFNTAD GFKYFAHHDE DLGNEEGEAL SYSGILNFNN KIYYFDDSFT AVVGW KDLE DGSKYYFDEN TAEASIGISI INDGKYYFND SGIMQIGFVT INNEVFYFSD SGIVESGMQN IDDNYFYISE NGLVQI GVF DTSDGYKYFA PANTVNDNIY GQAVEYSGLV RVNEDVYSFG ESYTIETGWI YDSENESDKY YFDPETKKAY KGINVID DI KYYFDENGIM RTGLITFEDN HYYFNEDGEM QYGYLNIEDK MFYFSEDGIM QIGVFNTPDG FKYFAHQNTL DENFEGES I NYTGWLDLDE KRYYFTDEYI AATGSVIIDG EEYYFDPDTA QLVISEHHHH HHHH

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Macromolecule #2: De novo design Minibinder

MacromoleculeName: De novo design Minibinder / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.43746 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
NEEEKFKFFV WFLAIRAGVP EVEVRNDNGK FQVTVKGDTD AARLLTKEVK EVATFLGVDV DLQIR

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration6 mg/mL
BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7v1n

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 228588
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8y9c:
De novo design mini-binder in complex with TcdB4

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