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- EMDB-48223: De novo designed minibinder complexed with Clostridioides diffici... -

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Basic information

Entry
Database: EMDB / ID: EMD-48223
TitleDe novo designed minibinder complexed with Clostridioides difficile Toxin B
Map dataMap of TcdB bound to CSPG4 site minibinder
Sample
  • Complex: De novo designed minibinder complexed with Clostridioides difficile Toxin B
    • Protein or peptide: Toxin B
    • Protein or peptide: De novo designed cspg67 minibinder
KeywordsTcdB / minibinder / de novo / inhibitor / TOXIN
Function / homology
Function and homology information


symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / 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 ...symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / 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, 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. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsMiletic S / Li Z / Ragotte RJ / Melnyk R
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)452580 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: De novo design of potent inhibitors of clostridial family toxins.
Authors: Robert J Ragotte / Huazhu Liang / John Tam / Sean Miletic / Jacob M Berman / Roger Palou / Connor Weidle / Zhijie Li / Matthias Glögl / Greg L Beilhartz / Kenneth D Carr / Andrew J Borst / ...Authors: Robert J Ragotte / Huazhu Liang / John Tam / Sean Miletic / Jacob M Berman / Roger Palou / Connor Weidle / Zhijie Li / Matthias Glögl / Greg L Beilhartz / Kenneth D Carr / Andrew J Borst / Brian Coventry / Xinru Wang / John L Rubinstein / Mike Tyers / Daniel Schramek / Roman A Melnyk / David Baker /
Abstract: remains a leading cause of hospital-acquired infections, with its primary virulence factor, toxin B (TcdB), responsible for severe colitis and recurrent disease. The closely related toxin, TcsL, ... remains a leading cause of hospital-acquired infections, with its primary virulence factor, toxin B (TcdB), responsible for severe colitis and recurrent disease. The closely related toxin, TcsL, from , causes a rarer but often fatal toxic shock syndrome, particularly in gynecological and obstetric contexts. We report the de novo design of small protein minibinders that directly neutralize TcdB and TcsL by preventing their entry into host cells. Using deep learning and Rosetta-based approaches, we generated high-affinity minibinders that protect cells from intoxication with picomolar potency and, in the case of TcsL, prolonged survival following lethal toxin challenge in mice. The designed proteins against TcdB demonstrate exceptional stability in proteolytic and acidic environments, making them well-suited for oral delivery-a valuable feature for treating infections localized to the gastrointestinal tract. For TcsL, potent inhibitors were identified from 48 initial designs and 48 optimized designs, highlighting the potential of computational design for rapidly developing countermeasures against life-threatening bacterial toxins.
History
DepositionDec 9, 2024-
Header (metadata) releaseSep 17, 2025-
Map releaseSep 17, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48223.png

Downloads & links

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Map

FileDownload / File: emd_48223.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of TcdB bound to CSPG4 site minibinder
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 320 pix.
= 329.6 Å		1.03 Å/pix.
x 320 pix.
= 329.6 Å		1.03 Å/pix.
x 320 pix.
= 329.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0432
Minimum - Maximum-0.087857015 - 0.24417086
Average (Standard dev.)0.00005466425 (±0.0043926286)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 329.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A of TcdB bound to CSPG4 site minibinder

Fileemd_48223_half_map_1.map
AnnotationHalf map A of TcdB bound to CSPG4 site minibinder
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of TcdB bound to CSPG4 site minibinder

Fileemd_48223_half_map_2.map
AnnotationHalf map B of TcdB bound to CSPG4 site minibinder
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : De novo designed minibinder complexed with Clostridioides diffici...

EntireName: De novo designed minibinder complexed with Clostridioides difficile Toxin B
Components
  • Complex: De novo designed minibinder complexed with Clostridioides difficile Toxin B
    • Protein or peptide: Toxin B
    • Protein or peptide: De novo designed cspg67 minibinder

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Supramolecule #1: De novo designed minibinder complexed with Clostridioides diffici...

SupramoleculeName: De novo designed minibinder complexed with Clostridioides difficile Toxin B
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridioides difficile (bacteria) / Strain: VPI 10463
Molecular weightTheoretical: 278 KDa

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Macromolecule #1: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Clostridioides difficile (bacteria) / Strain: VPI 10463
Molecular weightTheoretical: 270.767281 KDa
Recombinant expressionOrganism: Priestia megaterium (bacteria)
SequenceString: MSLVNRKQLE KMANVRFRTQ EDEYVAILDA LEEYHNMSEN TVVEKYLKLK DINSLTDIYI DTYKKSGRNK ALKKFKEYLV TEVLELKNN NLTPVEKNLH FVWIGGQIND TAINYINQWK DVNSDYNVNV FYDSNAFLIN TLKKTVVESA INDTLESFRE N LNDPRFDY ...String:
MSLVNRKQLE KMANVRFRTQ EDEYVAILDA LEEYHNMSEN TVVEKYLKLK DINSLTDIYI DTYKKSGRNK ALKKFKEYLV TEVLELKNN NLTPVEKNLH FVWIGGQIND TAINYINQWK DVNSDYNVNV FYDSNAFLIN TLKKTVVESA INDTLESFRE N LNDPRFDY NKFFRKRMEI IYDKQKNFIN YYKAQREENP ELIIDDIVKT YLSNEYSKEI DELNTYIEES LNKITQNSGN DV RNFEEFK NGESFNLYEQ ELVERWNLAA ASDILRISAL KEIGGMYLDV DMLPGIQPDL FESIEKPSSV TVDFWEMTKL EAI MKYKEY IPEYTSEHFD MLDEEVQSSF ESVLASKSDK SEIFSSLGDM EASPLEVKIA FNSKGIINQG LISVKDSYCS NLIV KQIEN RYKILNNSLN PAISEDNDFN TTTNTFIDSI MAEANADNGR FMMELGKYLR VGFFPDVKTT INLSGPEAYA AAYQD LLMF KEGSMNIHLI EADLRNFEIS KTNISQSTEQ EMASLWSFDD ARAKAQFEEY KRNYFEGSLG EDDNLDFSQN IVVDKE YLL EKISSLARSS ERGYIHYIVQ LQGDKISYEA ACNLFAKTPY DSVLFQKNIE DSEIAYYYNP GDGEIQEIDK YKIPSII SD RPKIKLTFIG HGKDEFNTDI FAGFDVDSLS TEIEAAIDLA KEDISPKSIE INLLGCNMFS YSINVEETYP GKLLLKVK D KISELMPSIS QDSIIVSANQ YEVRINSEGR RELLDHSGEW INKEESIIKD ISSKEYISFN PKENKITVKS KNLPELSTL LQEIRNNSNS SDIELEEKVM LTECEINVIS NIDTQIVEER IEEAKNLTSD SINYIKDEFK LIESISDALC DLKQQNELED SHFISFEDI SETDEGFSIR FINKETGESI FVETEKTIFS EYANHITEEI SKIKGTIFDT VNGKLVKKVN LDTTHEVNTL N AAFFIQSL IEYNSSKESL SNLSVAMKVQ VYAQLFSTGL NTITDAAKVV ELVSTALDET IDLLPTLSEG LPIIATIIDG VS LGAAIKE LSETSDPLLR QEIEAKIGIM AVNLTTATTA IITSSLGIAS GFSILLVPLA GISAGIPSLV NNELVLRDKA TKV VDYFKH VSLVETEGVF TLLDDKIMMP QDDLVISEID FNNNSIVLGK CEIWRMEGGS GHTVTDDIDH FFSAPSITYR EPHL SIYDV LEVQKEELDL SKDLMVLPNA PNRVFAWETG WTPGLRSLEN DGTKLLDRIR DNYEGEFYWR YFAFIADALI TTLKP RYED TNIRINLDSN TRSFIVPIIT TEYIREKLSY SFYGSGGTYA LSLSQYNMGI NIELSESDVW IIDVDNVVRD VTIESD KIK KGDLIEGILS TLSIEENKII LNSHEINFSG EVNGSNGFVS LTFSILEGIN AIIEVDLLSK SYKLLISGEL KILMLNS NH IQQKIDYIGF NSELQKNIPY SFVDSEGKEN GFINGSTKEG LFVSELPDVV LISKVYMDDS KPSFGYYSNN LKDVKVIT K DNVNILTGYY LKDDIKISLS LTLQDEKTIK LNSVHLDESG VAEILKFMNR KGNTNTSDSL MSFLESMNIK SIFVNFLQS NIKFILDANF IISGTTSIGQ FEFICDENDN IQPYFIKFNT LETNYTLYVG NRQNMIVEPN YDLDDSGDIS STVINFSQKY LYGIDSCVN KVVISPNIYT DEINITPVYE TNNTYPEVIV LDANYINEKI NVNINDLSIR YVWSNDGNDF ILMSTSEENK V SQVKIRFV NVFKDKTLAN KLSFNFSDKQ DVPVSEIILS FTPSYYEDGL IGYDLGLVSL YNEKFYINNF GMMVSGLIYI ND SLYYFKP PVNNLITGFV TVGDDKYYFN PINGGAASIG ETIIDDKNYY FNQSGVLQTG VFSTEDGFKY FAPANTLDEN LEG EAIDFT GKLIIDENIY YFDDNYRGAV EWKELDGEMH YFSPETGKAF KGLNQIGDYK YYFNSDGVMQ KGFVSINDNK HYFD DSGVM KVGYTEIDGK HFYFAENGEM QIGVFNTEDG FKYFAHHNED LGNEEGEEIS YSGILNFNNK IYYFDDSFTA VVGWK DLED GSKYYFDEDT AEAYIGLSLI NDGQYYFNDD GIMQVGFVTI NDKVFYFSDS GIIESGVQNI DDNYFYIDDN GIVQIG VFD TSDGYKYFAP ANTVNDNIYG QAVEYSGLVR VGEDVYYFGE TYTIETGWIY DMENESDKYY FNPETKKACK GINLIDD IK YYFDEKGIMR TGLISFENNN YYFNENGEMQ FGYINIEDKM FYFGEDGVMQ IGVFNTPDGF KYFAHQNTLD ENFEGESI N YTGWLDLDEK RYYFTDEYIA ATGSVIIDGE EYYFDPDTAQ LVISEHHHHH H

UniProtKB: Toxin B

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Macromolecule #2: De novo designed cspg67 minibinder

MacromoleculeName: De novo designed cspg67 minibinder / type: protein_or_peptide / ID: 2
Details: De novo designed mini protein inhibitor (minibinder) targeting CSPG4 receptor binding site on TcdB
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.640785 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
AEELAKKLEE MAEVFKEKGD EEGAKECLKA AKIVRRSKNE EFGKWCIKKA EERLKKL

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMNaClsodium chloride
20.0 mMtris
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 4.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: 30s preblot incubation on grid. 2-3s blot time..
DetailsPurified TcdB was mixed with CSPG4 minibinder #67 at a 1:1 molar ratio and incubated on ice until plunge freezing.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 42.0 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: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 179414
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4)

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Atomic model buiding 1

Initial modelPDB ID:

8177


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsPredicted models were docked into the map using ChimeraX. The model was then manually with ISOLDE and refined with phenix.real_space_refine.
RefinementProtocol: OTHER / Overall B value: 113
Output model

PDB-9mf4:
De novo designed minibinder complexed with Clostridioides difficile Toxin B

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