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- EMDB-18373: cryo-EM structure of apo Clostridioides difficile toxin B -

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Basic information

Entry
Database: EMDB / ID: EMD-18373
Titlecryo-EM structure of apo Clostridioides difficile toxin B
Map dataoverall map used for defining masked regions
Sample
  • Organelle or cellular component: apo-structure of Clostridioides difficile toxin B
    • Organelle or cellular component: Masked region 1
      • Protein or peptide: Toxin B
    • Organelle or cellular component: Masked region 2
  • Ligand: ZINC ION
Keywordsmicrobiology / pore forming region / CROP dynamics / TOXIN / Structural Genomics / Structural Genomics Consortium / SGC
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 / membrane / metal ion binding
Similarity search - Function
Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Membrane Localization 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 ...Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Membrane Localization 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 speciesPriestia megaterium DSM 319 (bacteria) / Clostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKinsolving J / Bous J / Structural Genomics Consortium (SGC)
Funding support Sweden, Denmark, 11 items
OrganizationGrant numberCountry
Swedish Research Council2019-01190 Sweden
Cancerfonden20 1102 PjF Sweden
Novo Nordisk FoundationNNF21OC0070008 Denmark
Novo Nordisk FoundationNNF22OC0078104 Denmark
Other government2021-00430
Other government2018-01562
Swedish Research Council2022-03681 Sweden
Swedish Research Council2018-03406 Sweden
Cancerfonden20 1287 PjF Sweden
Cancerfonden202 0264 PjF Sweden
Swedish Research Council2022-01398 Sweden
CitationJournal: Cell Rep / Year: 2024
Title: Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD.
Authors: Julia Kinsolving / Julien Bous / Pawel Kozielewicz / Sara Košenina / Rawan Shekhani / Lukas Grätz / Geoffrey Masuyer / Yuankai Wang / Pål Stenmark / Min Dong / Gunnar Schulte /
Abstract: The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible ...The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
History
DepositionSep 1, 2023-
Header (metadata) releaseMar 20, 2024-
Map releaseMar 20, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18373.png

Downloads & links

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Map

FileDownload / File: emd_18373.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationoverall map used for defining masked regions
Voxel sizeX=Y=Z: 1.1592 Å
Density
Contour LevelBy AUTHOR: 0.504
Minimum - Maximum-0.4411072 - 2.1878943
Average (Standard dev.)0.015436061 (±0.041844785)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 579.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18373_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Mask #2

Fileemd_18373_msk_2.map
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Half map: #1

Fileemd_18373_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_18373_half_map_2.map
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Sample components

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Entire : apo-structure of Clostridioides difficile toxin B

EntireName: apo-structure of Clostridioides difficile toxin B
Components
  • Organelle or cellular component: apo-structure of Clostridioides difficile toxin B
    • Organelle or cellular component: Masked region 1
      • Protein or peptide: Toxin B
    • Organelle or cellular component: Masked region 2
  • Ligand: ZINC ION

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Supramolecule #1: apo-structure of Clostridioides difficile toxin B

SupramoleculeName: apo-structure of Clostridioides difficile toxin B / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Priestia megaterium DSM 319 (bacteria)

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Supramolecule #2: Masked region 1

SupramoleculeName: Masked region 1 / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Priestia megaterium DSM 319 (bacteria)

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Supramolecule #3: Masked region 2

SupramoleculeName: Masked region 2 / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Priestia megaterium DSM 319 (bacteria)

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Macromolecule #1: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 273.551562 KDa
Recombinant expressionOrganism: Priestia megaterium DSM 319 (bacteria)
SequenceString: MDKLVHLNQR GKCTMSLVNR KQLEKMANVR FRTQEDEYVA ILDALEEYHN MSENTVVEKY LKLKDINSLT DIYIDTYKKS GRNKALKKF KEYLVTEVLE LKNNNLTPVE KNLHFVWIGG QINDTAINYI NQWKDVNSDY NVNVFYDSNA FLINTLKKTV V ESAINDTL ...String:
MDKLVHLNQR GKCTMSLVNR KQLEKMANVR FRTQEDEYVA ILDALEEYHN MSENTVVEKY LKLKDINSLT DIYIDTYKKS GRNKALKKF KEYLVTEVLE LKNNNLTPVE KNLHFVWIGG QINDTAINYI NQWKDVNSDY NVNVFYDSNA FLINTLKKTV V ESAINDTL ESFRENLNDP RFDYNKFFRK RMEIIYDKQK NFINYYKAQR EENPELIIDD IVKTYLSNEY SKEIDELNTY IE ESLNKIT QNSGNDVRNF EEFKNGESFN LYEQELVERW NLAAASDILR ISALKEIGGM YLDVDMLPGI QPDLFESIEK PSS VTVDFW EMTKLEAIMK YKEYIPEYTS EHFDMLDEEV QSSFESVLAS KSDKSEIFSS LGDMEASPLE VKIAFNSKGI INQG LISVK DSYCSNLIVK QIENRYKILN NSLNPAISED NDFNTTTNTF IDSIMAEANA DNGRFMMELG KYLRVGFFPD VKTTI NLSG PEAYAAAYQD LLMFKEGSMN IHLIEADLRN FEISKTNISQ STEQEMASLW SFDDARAKAQ FEEYKRNYFE GSLGED DNL DFSQNIVVDK EYLLEKISSL ARSSERGYIH YIVQLQGDKI SYEAACNLFA KTPYDSVLFQ KNIEDSEIAY YYNPGDG EI QEIDKYKIPS IISDRPKIKL TFIGHGKDEF NTDIFAGFDV DSLSTEIEAA IDLAKEDISP KSIEINLLGC NMFSYSIN V EETYPGKLLL KVKDKISELM PSISQDSIIV SANQYEVRIN SEGRRELLDH SGEWINKEES IIKDISSKEY ISFNPKENK ITVKSKNLPE LSTLLQEIRN NSNSSDIELE EKVMLTECEI NVISNIDTQI VEERIEEAKN LTSDSINYIK DEFKLIESIS DALCDLKQQ NELEDSHFIS FEDISETDEG FSIRFINKET GESIFVETEK TIFSEYANHI TEEISKIKGT IFDTVNGKLV K KVNLDTTH EVNTLNAAFF IQSLIEYNSS KESLSNLSVA MKVQVYAQLF STGLNTITDA AKVVELVSTA LDETIDLLPT LS EGLPIIA TIIDGVSLGA AIKELSETSD PLLRQEIEAK IGIMAVNLTT ATTAIITSSL GIASGFSILL VPLAGISAGI PSL VNNELV LRDKATKVVD YFKHVSLVET EGVFTLLDDK IMMPQDDLVI SEIDFNNNSI VLGKCEIWRM EGGSGHTVTD DIDH FFSAP SITYREPHLS IYDVLEVQKE ELDLSKDLMV LPNAPNRVFA WETGWTPGLR SLENDGTKLL DRIRDNYEGE FYWRY FAFI ADALITTLKP RYEDTNIRIN LDSNTRSFIV PIITTEYIRE KLSYSFYGSG GTYALSLSQY NMGINIELSE SDVWII DVD NVVRDVTIES DKIKKGDLIE GILSTLSIEE NKIILNSHEI NFSGEVNGSN GFVSLTFSIL EGINAIIEVD LLSKSYK LL ISGELKILML NSNHIQQKID YIGFNSELQK NIPYSFVDSE GKENGFINGS TKEGLFVSEL PDVVLISKVY MDDSKPSF G YYSNNLKDVK VITKDNVNIL TGYYLKDDIK ISLSLTLQDE KTIKLNSVHL DESGVAEILK FMNRKGNTNT SDSLMSFLE SMNIKSIFVN FLQSNIKFIL DANFIISGTT SIGQFEFICD ENDNIQPYFI KFNTLETNYT LYVGNRQNMI VEPNYDLDDS GDISSTVIN FSQKYLYGID SCVNKVVISP NIYTDEINIT PVYETNNTYP EVIVLDANYI NEKINVNIND LSIRYVWSND G NDFILMST SEENKVSQVK IRFVNVFKDK TLANKLSFNF SDKQDVPVSE IILSFTPSYY EDGLIGYDLG LVSLYNEKFY IN NFGMMVS GLIYINDSLY YFKPPVNNLI TGFVTVGDDK YYFNPINGGA ASIGETIIDD KNYYFNQSGV LQTGVFSTED GFK YFAPAN TLDENLEGEA IDFTGKLIID ENIYYFDDNY RGAVEWKELD GEMHYFSPET GKAFKGLNQI GDYKYYFNSD GVMQ KGFVS INDNKHYFDD SGVMKVGYTE IDGKHFYFAE NGEMQIGVFN TEDGFKYFAH HNEDLGNEEG EEISYSGILN FNNKI YYFD DSFTAVVGWK DLEDGSKYYF DEDTAEAYIG LSLINDGQYY FNDDGIMQVG FVTINDKVFY FSDSGIIESG VQNIDD NYF YIDDNGIVQI GVFDTSDGYK YFAPANTVND NIYGQAVEYS GLVRVGEDVY YFGETYTIET GWIYDMENES DKYYFNP ET KKACKGINLI DDIKYYFDEK GIMRTGLISF ENNNYYFNEN GEMQFGYINI EDKMFYFGED GVMQIGVFNT PDGFKYFA H QNTLDENFEG ESINYTGWLD LDEKRYYFTD EYIAATGSVI IDGEEYYFDP DTAQLVISEG YRPHAGLRGS HHHHHH

UniProtKB: Toxin B

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / 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

Concentration0.630 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.1 MTRIS-HClTristris hydrochloride
0.2 MNaClSodium chloridesodium chloride
0.002 %LMNGlauryl maltose neopentyl glycol
0.0002 %CHScholesteryl hemisuccinate tris salt
0.0002 %GDNglyco diosgenin

Details: 100 mM TRIS-HCl pH 7.5 200 mM NaCl 0.002% LMNG 0.0002% CHS 0.0002% GDN
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.101 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 17269 / Average exposure time: 3.0 sec. / Average electron dose: 50.453 e/Å2
Details: Images collected in super-resolution mode, faster acquisition mode, with 4 exposures per hole
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5240176
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 100000 / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 41523
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8qen:
cryo-EM structure of apo Clostridioides difficile toxin B

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