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- EMDB-44419: Structure of Clostridioides difficile Component A (50-463) in Com... -

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Basic information

Entry
Database: EMDB / ID: EMD-44419
TitleStructure of Clostridioides difficile Component A (50-463) in Complex with a CDTb Oligomer
Map data
Sample
  • Complex: Clostridioides difficile Transferase component A in complex with Clostridioides difficile Transferase component B
    • Protein or peptide: ADP-ribosyltransferase binding component
    • Protein or peptide: ADP-ribosyltransferase enzymatic component
  • Ligand: CALCIUM ION
KeywordsClostridioides / difficile / toxin / CDT / TRANSFERASE
Function / homology
Function and homology information


Transferases; Glycosyltransferases; Pentosyltransferases / protein homooligomerization / extracellular region / identical protein binding
Similarity search - Function
Binary exotoxin A, clostridial type / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / Bacterial exotoxin B / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily / Clostridial binary toxin B/anthrax toxin PA Ca-binding domain ...Binary exotoxin A, clostridial type / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / Bacterial exotoxin B / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily / Clostridial binary toxin B/anthrax toxin PA Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA domain 2 / Clostridial binary toxin B/anthrax toxin PA domain 3 / PA14 domain / PA14 / PA14 domain
Similarity search - Domain/homology
ADP-ribosyltransferase binding component / ADP-ribosyltransferase enzymatic component
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSheedlo MJ / Mullard RM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R00AI154672 United States
CitationJournal: mBio / Year: 2025
Title: The N-terminus of the transferase A component directs toxin activity and potency.
Authors: Robin M Mullard / Michael J Sheedlo /
Abstract: infection is the leading cause of antibiotic-associated, hospital-acquired diarrhea in the USA; the pathology of which is mediated by toxins. The presence of a toxin known as the Transferase (CDT) ... infection is the leading cause of antibiotic-associated, hospital-acquired diarrhea in the USA; the pathology of which is mediated by toxins. The presence of a toxin known as the Transferase (CDT) in some clinical isolates is linked to severe symptoms including increased incidence of reinfection and higher rates of mortality. Despite its apparent importance to pathology, a mechanistic model of how CDT intoxicates cells remains incomplete. Here, we describe a motif composed of acidic and basic residues (the KDKEK motif) that is essential for toxin function. Using Cryogenic Electron Microscopy (Cryo-EM), we highlight an orientation of the KDKEK motif wherein the acidic residues engage structures thought to play an important role during toxin delivery. We thus present a model wherein these interactions prime CDT for entry into host cells. We expect that this model can be extrapolated to other bacterial toxins to understand how they enter cells.IMPORTANCE is the leading cause of hospital-acquired infectious diarrhea in the USA. The pathology that accompanies infection is triggered by toxins produced by the bacterium. One of these, the Transferase (CDT), has been associated with poorer patient outcomes, although a direct connection to CDT activity has remained elusive. Herein, we present new insight into the mechanism of CDT intoxication and define two regions of the toxin as important for its activity. Moreover, we have generated mutants of CDT that retain the ability to assemble but can no longer intoxicate host cells. In the future, we expect these mutants will serve as valuable tools to help elucidate the role of CDT during infection.
History
DepositionApr 5, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateJun 10, 2026-
Current statusJun 10, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44419.png

Downloads & links

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Map

FileDownload / File: emd_44419.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 340 pix.
= 221. Å		0.65 Å/pix.
x 340 pix.
= 221. Å		0.65 Å/pix.
x 340 pix.
= 221. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.0540575 - 1.7252381
Average (Standard dev.)0.0055412534 (±0.06410685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 220.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44419_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44419_half_map_2.map
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 Transferase component A in complex with ...

EntireName: Clostridioides difficile Transferase component A in complex with Clostridioides difficile Transferase component B
Components
  • Complex: Clostridioides difficile Transferase component A in complex with Clostridioides difficile Transferase component B
    • Protein or peptide: ADP-ribosyltransferase binding component
    • Protein or peptide: ADP-ribosyltransferase enzymatic component
  • Ligand: CALCIUM ION

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Supramolecule #1: Clostridioides difficile Transferase component A in complex with ...

SupramoleculeName: Clostridioides difficile Transferase component A in complex with Clostridioides difficile Transferase component B
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Clostridioides difficile (bacteria)

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Macromolecule #1: ADP-ribosyltransferase binding component

MacromoleculeName: ADP-ribosyltransferase binding component / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 94.289367 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEIVNEDILP NNGLMGYYFT DEHFKDLKLM APIKDGNLKF EEKKVDKLLD KDKSDVKSIR WTGRIIPSKD GEYTLSTDRD DVLMQVNTE STISNTLKVN MKKGKEYKVR IELQDKNLGS IDNLSSPNLY WELDGMKKII PEENLFLRDY SNIEKDDPFI P NNNFFDPK ...String:
MEIVNEDILP NNGLMGYYFT DEHFKDLKLM APIKDGNLKF EEKKVDKLLD KDKSDVKSIR WTGRIIPSKD GEYTLSTDRD DVLMQVNTE STISNTLKVN MKKGKEYKVR IELQDKNLGS IDNLSSPNLY WELDGMKKII PEENLFLRDY SNIEKDDPFI P NNNFFDPK LMSDWEDEDL DTDNDNIPDS YERNGYTIKD LIAVKWEDSF AEQGYKKYVS NYLESNTAGD PYTDYEKASG SF DKAIKTE ARDPLVAAYP IVGVGMEKLI ISTNEHASTD QGKTVSRATT NSKTESNTAG VSVNVGYQNG FTANVTTNYS HTT DNSTAV QDSNGESWNT GLSINKGESA YINANVRYYN TGTAPMYKVT PTTNLVLDGD TLSTIKAQEN QIGNNLSPGD TYPK KGLSP LALNTMDQFS SRLIPINYDQ LKKLDAGKQI KLETTQVSGN FGTKNSSGQI VTEGNSWSDY ISQIDSISAS IILDT ENES YERRVTAKNL QDPEDKTPEL TIGEAIEKAF GATKKDGLLY FNDIPIDESC VELIFDDNTA NKIKDSLKTL SDKKIY NVK LERGMNILIK TPTYFTNFDD YNNYPSTWSN VNTTNQDGLQ GSANKLNGET KIKIPMSELK PYKRYVFSGY SKDPLTS NS IIVKIKAKEE KTDYLVPEQG YTKFSYEFET TEKDSSNIEI TLIGSGTTYL DNLSITELNS TPEILDEPEV KIPTDQEI M DAHKIYFADL NFNPSTGNTY INGMYFAPTQ TNKEALDYIQ KYRVEATLQY SGFKDIGTKD KEMRNYLGDP NQPKTNYVN LRSYFTGGEN IMTYKKLRIY AITPDDRELL VLSVD

UniProtKB: ADP-ribosyltransferase binding component

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Macromolecule #2: ADP-ribosyltransferase enzymatic component

MacromoleculeName: ADP-ribosyltransferase enzymatic component / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 47.626613 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KAPIERPEDF LKDKEKAKEW ERKEAERIEQ KLERSEKEAL ESYKKDSVEI SKYSQTRNYF YDYQIEANSR EKEYKELRNA ISKNKIDKP MYVYYFESPE KFAFNKVIRT ENQNEISLEK FNEFKETIQN KLFKQDGFKD ISLYEPGKGD EKPTPLLMHL K LPRNTGML ...String:
KAPIERPEDF LKDKEKAKEW ERKEAERIEQ KLERSEKEAL ESYKKDSVEI SKYSQTRNYF YDYQIEANSR EKEYKELRNA ISKNKIDKP MYVYYFESPE KFAFNKVIRT ENQNEISLEK FNEFKETIQN KLFKQDGFKD ISLYEPGKGD EKPTPLLMHL K LPRNTGML PYTNTNNVST LIEQGYSIKI DKIVRIVIDG KHYIKAEASV VSSLDFKDDV SKGDSWGKAN YNDWSNKLTP NE LADVNDY MRGGYTAINN YLISNGPVNN PNPELDSKIT NIENALKREP IPTNLTVYRR SGPQEFGLTL TSPEYDFNKL ENI DAFKSK WEGQALSYPN FISTSIGSVN MSAFAKRKIV LRITIPKGSP GAYLSAIPGY AGEYEVLLNH GSKFKINKID SYKD GTITK LIVDATLIP

UniProtKB: ADP-ribosyltransferase enzymatic component

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 14 / Formula: CA
Molecular weightTheoretical: 40.078 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

BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.2.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 43726
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)

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