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- EMDB-45739: CryoEM Strucuture of TcdB in complex with De Novo Minibinder fzd48 -

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Basic information

Entry
Database: EMDB / ID: EMD-45739
TitleCryoEM Strucuture of TcdB in complex with De Novo Minibinder fzd48
Map dataDeepEMhancer Map, Main Map
Sample
  • Complex: tcdB with de novo minibinder fzd48
    • Protein or peptide: De Novo Minibinder fzd48
    • Protein or peptide: Toxin B
KeywordsTcdB / Clostridium difficile / C. Diff. / fzd48 / De Novo Minibinder / TOXIN
Function / homology
Function and homology information


symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / glucosyltransferase activity / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / 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 / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / 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 speciesEscherichia coli (E. coli) / synthetic construct (others) / Clostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.61 Å
AuthorsWeidle C / Carr KD / Borst AJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
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
DepositionJul 12, 2024-
Header (metadata) releaseMay 21, 2025-
Map releaseMay 21, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45739.png

Downloads & links

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Map

FileDownload / File: emd_45739.map.gz / Format: CCP4 / Size: 371.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer Map, Main Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 460 pix.
= 387.78 Å		0.84 Å/pix.
x 460 pix.
= 387.78 Å		0.84 Å/pix.
x 460 pix.
= 387.78 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.843 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0803
Minimum - Maximum-0.001755648 - 2.4823716
Average (Standard dev.)0.00078535127 (±0.022123175)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions460460460
Spacing460460460
CellA=B=C: 387.78 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Non Uniform Refinement Map

Fileemd_45739_additional_1.map
AnnotationNon Uniform Refinement Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non Uniform Refinement Sharpened Map

Fileemd_45739_additional_2.map
AnnotationNon Uniform Refinement Sharpened Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Non Uniform Refinement, Half Map B

Fileemd_45739_half_map_1.map
AnnotationNon Uniform Refinement, Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Non Uniform Refinement, Half Map A

Fileemd_45739_half_map_2.map
AnnotationNon Uniform Refinement, Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : tcdB with de novo minibinder fzd48

EntireName: tcdB with de novo minibinder fzd48
Components
  • Complex: tcdB with de novo minibinder fzd48
    • Protein or peptide: De Novo Minibinder fzd48
    • Protein or peptide: Toxin B

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Supramolecule #1: tcdB with de novo minibinder fzd48

SupramoleculeName: tcdB with de novo minibinder fzd48 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: tcdB and minibinder were mixed at 1:3 molar ratio 10 minutes before freezing.
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 249.89658 KDa

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Macromolecule #1: De Novo Minibinder fzd48

MacromoleculeName: De Novo Minibinder fzd48 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.926099 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MSGKEELEKM IEELKKLIEN GDKENFIKLF DEAFKKAKES RDPRTIASVW TLVLEFKNKN GSGSHHWGST HHHHHH

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Macromolecule #2: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 241.229938 KDa
Recombinant expressionOrganism: Priestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
SequenceString: MYMSLVNRKQ LEKMANVRFR TQEDEYVAIL DALEEYHNMS ENTVVEKYLK LKDINSLTDI YIDTYKKSGR NKALKKFKEY LVTEVLELK NNNLTPVEKN LHFVAIGGQI NDTAINYINQ WKDVNSDYNV NVFYDSNAFL INTLKKTVVE SAINDTLESF R ENLNDPRF ...String:
MYMSLVNRKQ LEKMANVRFR TQEDEYVAIL DALEEYHNMS ENTVVEKYLK LKDINSLTDI YIDTYKKSGR NKALKKFKEY LVTEVLELK NNNLTPVEKN LHFVAIGGQI NDTAINYINQ WKDVNSDYNV NVFYDSNAFL INTLKKTVVE SAINDTLESF R ENLNDPRF DYNKFFRKRM EIIYDKQKNF INYYKAQREE NPELIIDDIV KTYLSNEYSK EIDELNTYIE ESLNKITQNS GN DVRNFEE FKNGESFNLY EQELVERWNL AAASDILRIS ALKEIGGMYL NVNMLPGIQP DLFESIEKPS SVTVDFWEMT KLE AIMKYK EYIPEYTSEH FDMLDEEVQS SFESVLASKS DKSEIFSSLG DMEASPLEVK IAFNSKGIIN QGLISVKDSY CSNL IVKQI ENRYKILNNS LNPAISEDND FNTTTNTFID SIMAEANADN GRFMMELGKY LRVGFFPDVK TTINLSGPEA YAAAY QDLL MFKEGSMNIH LIEADLRNFE ISKTNISQST EQEMASLWSF DDARAKAQFE EYKRNYFEGS AGEDDNLDFS QNIVVD KEY LLEKISSLAR SSERGYIHYI VQLQGDKISY EAACNLFAKT PYDSVLFQKN IEDSEIAYYY NPGDGEIQEI DKYKIPS II SDRPKIKLTF IGHGKDEFNT DIFAGFDVDS LSTEIEAAID LAKEDISPKS IEINLLGCNM FSYSINVEET YPGKLLLK V KDKISELMPS ISQDSIIVSA NQYEVRINSE GRRELLDHSG EWINKEESII KDISSKEYIS FNPKENKITV KSKNLPELS TLLQEIRNNS NSSDIELEEK VMLTECEINV ISNIDTQIVE ERIEEAKNLT SDSINYIKDE FKLIESISDA LCDLKQQNEL EDSHFISFE DISETDEGFS IRFINKETGE SIFVETEKTI FSEYANHITE EISKIKGTIF DTVNGKLVKK VNLDTTHEVN T LNAAFFIQ SLIEYNSSKE SLSNLSVAMK VQVYAQLFST GLNTITDAAK VVELVSTALD ETIDLLPTLS EGLPIIATII DG VSLGAAI KELSETSDPL LRQEIEAKIG IMAVNLTTAT TAIITSSLGI ASGFSILLVP LAGISAGIPS LVNNELVLRD KAT KVVDYF KHVSLVETEG VFTLLDDKIM MPQDDLVISE IDFNNNSIVL GKCEIWRMEG GSGHTVTDDI DHFFSAPSIT YREP HLSIY DVLEVQKEEL DLSKDLMVLP NAPNRVFAWE TGWTPGLRSL ENDGTKLLDR IRDNYEGEFY WRYFAFIADA LITTL KPRY EDTNIRINLD SNTRSFIVPI ITTEYIREKL SYSFYGSGGT YALSLSQYNM GINIELSESD VWIIDVDNVV RDVTIE SDK IKKGDLIEGI LSTLSIEENK IILNSHEINF SGEVNGSNGF VSLTFSILEG INAIIEVDLL SKSYKLLISG ELKILML NS NHIQQKIDYI GFNSELQKNI PYSFVDSEGK ENGFINGSTK EGLFVSELPD VVLISKVYMD DSKPSFGYYS NNLKDVKV I TKDNVNILTG YYLKDDIKIS LSLTLQDEKT IKLNSVHLDE SGVAEILKFM NRKGNTNTSD SLMSFLESMN IKSIFVNFL QSNIKFILDA NFIISGTTSI GQFEFICDEN DNIQPYFIKF NTLETNYTLY VGNRQNMIVE PNYDLDDSGD ISSTVINFSQ KYLYGIDSC VNKVVISPNI YTDEINITPV YETNNTYPEV IVLDANYINE KINVNINDLS IRYVWSNDGN DFILMSTSEE N KVSQVKIR FVNVFKDKTL ANKLSFNFSD KQDVPVSEII LSFTPSYYED GLIGYDLGLV SLYNEKFYIN NFGMMVSGLI YI NDSLYYF KPPVNNLITG FVTVGDDKYY FNPINGGAAS IGETIIDDKN YYFNQSGVLQ TGVFSTEDGF KYFAPANTLD ENL EGEAID FTGKLIIDEN IYYFDDNYRG AVEWKELDGE MHYFSPETGK AFKGLNQIGD YKYYFNSDGV MQKGFVSIND NKHY FDDSG VMKVGYTEID GKHFYFAENG EMQIGVFNTE DGFKYFAHHN EDLGNEEGEE ISYSGILNFN NKIYYFDDSF TAVVG WKDL EDGSKYYFDE DTAEAYIGGY RPHAGLRGSH HHHHH

UniProtKB: Toxin B

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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

Concentration0.81 mg/mL
BufferpH: 7.5
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 39.0 kPa / Details: - 15mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was monodisperse, screened by negative stain prior to freezing

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6766 / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
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 correctionSoftware - Name: cryoSPARC / Software - details: PatchCTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Ab Initio
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.61 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 108076
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 23 / Avg.num./class: 4699 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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