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- EMDB-70486: CryoEM structure of Toxin B (TcdB) from clostridioides difficile ... -

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Basic information

Entry
Database: EMDB / ID: EMD-70486
TitleCryoEM structure of Toxin B (TcdB) from clostridioides difficile complexed with taurochenodeoxycholic acid (TCDCA)
Map datacryoEM map of Toxin B (TcdB) from Clostridioides difficile complexed with the natural bile acid TCDCA.
Sample
  • Complex: Clostridioides difficile Toxin B (TcdB) complexed with the natural bile acid TCDCA
    • Protein or peptide: Toxin B
  • Ligand: TAUROCHENODEOXYCHOLIC ACID
Keywordsclostridioides difficile / Toxin B / TcdB / Bile acid / TCDCA / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsMiletic S / Li Z / Melnyk RA
Funding support Canada, 1 items
OrganizationGrant numberCountry
The Hospital For Sick Children Foundation Canada
CitationJournal: Nat Microbiol / Year: 2025
Title: Structure-guided design of a synthetic bile acid that inhibits Clostridioides difficile TcdB toxin.
Authors: Sean Miletic / Simoun Icho / Zhijie Li / John Tam / Elizabeth C Rose / Cypress E Perkins / Ali Nakhi / Xinglin Yang / Howard C Hang / John L Rubinstein / Peter I Dosa / Casey M Theriot / Roman A Melnyk /
Abstract: Intestinal bile acids are a family of host and microbiota metabolites that can directly inhibit toxin B (TcdB), the primary virulence factor of Clostridioides difficile that causes infectious ...Intestinal bile acids are a family of host and microbiota metabolites that can directly inhibit toxin B (TcdB), the primary virulence factor of Clostridioides difficile that causes infectious diarrhoea and colitis. However, the mechanism underlying the inhibition is unclear. Here we used cryogenic electron microscopy and determined the structure of TcdB bound to inhibitory bile acids cholic acid (methyl ester) and taurochenodeoxycholic acid at 2.9 Å and 3.3 Å resolution, respectively. These structures revealed that bile acids lock the C-terminal CROP domain of TcdB in a conformation that allosterically masks the two receptor-binding sites and prevents target cell recognition. Guided by the structure, we synthesized gut-restricted bile acid derivatives, designed to evade the bile acid reuptake transporters within the gut. One of the derivatives, sBA-2, was retained within the gut upon oral dosing and protected mice from toxin-induced C. difficile disease pathology. Our study uncovers the structural basis of inhibition of TcdB by bile acids and its analogues, paving the way for the development of orally deliverable therapeutics against C. difficile.
History
DepositionMay 4, 2025-
Header (metadata) releaseMar 11, 2026-
Map releaseMar 11, 2026-
UpdateMar 11, 2026-
Current statusMar 11, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70486.png

Downloads & links

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Map

FileDownload / File: emd_70486.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM map of Toxin B (TcdB) from Clostridioides difficile complexed with the natural bile acid TCDCA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 384 pix.
= 395.52 Å		1.03 Å/pix.
x 384 pix.
= 395.52 Å		1.03 Å/pix.
x 384 pix.
= 395.52 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: 0.107
Minimum - Maximum-0.30634803 - 0.6468575
Average (Standard dev.)-0.00008384371 (±0.012200308)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 395.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A of Toxin B (TcdB) from...

Fileemd_70486_half_map_1.map
AnnotationHalf map A of Toxin B (TcdB) from Clostridioides difficile complexed with the natural bile acid TCDCA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of Toxin B (TcdB) from...

Fileemd_70486_half_map_2.map
AnnotationHalf map B of Toxin B (TcdB) from Clostridioides difficile complexed with the natural bile acid TCDCA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Clostridioides difficile Toxin B (TcdB) complexed with the natura...

EntireName: Clostridioides difficile Toxin B (TcdB) complexed with the natural bile acid TCDCA
Components
  • Complex: Clostridioides difficile Toxin B (TcdB) complexed with the natural bile acid TCDCA
    • Protein or peptide: Toxin B
  • Ligand: TAUROCHENODEOXYCHOLIC ACID

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Supramolecule #1: Clostridioides difficile Toxin B (TcdB) complexed with the natura...

SupramoleculeName: Clostridioides difficile Toxin B (TcdB) complexed with the natural bile acid TCDCA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: 500 micromolar TCDCA
Source (natural)Organism: Clostridioides difficile (bacteria) / Strain: VPI 10463
Molecular weightTheoretical: 276 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: TAUROCHENODEOXYCHOLIC ACID

MacromoleculeName: TAUROCHENODEOXYCHOLIC ACID / type: ligand / ID: 2 / Number of copies: 3 / Formula: TUD
Molecular weightTheoretical: 499.704 Da
Chemical component information

ChemComp-TUD:
TAUROCHENODEOXYCHOLIC ACID

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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

Details: 500 micromolar of TCDCA was added before freezing.
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 500 micromolar of TCDCA (4% DMSO final concentration) 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: 40.0 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: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 351161
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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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 built with ISOLDE and Coot and refined with phenix.real_space_refine.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9ohc:
CryoEM structure of Toxin B (TcdB) from clostridioides difficile complexed with taurochenodeoxycholic acid (TCDCA)

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