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- EMDB-21016: Structure of the Clostridioides difficile transferase toxin -

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Basic information

Entry
Database: EMDB / ID: EMD-21016
TitleStructure of the Clostridioides difficile transferase toxin
Map dataCryo-EM Reconstruction of CDT
Sample
  • Complex: C. difficile transferase toxin
    • Protein or peptide: ADP-ribosyltransferase binding componentPoly (ADP-ribose) polymerase
    • Protein or peptide: ADP-ribosylating binary toxin enzymatic subunit CdtA
  • Ligand: CALCIUM IONCalcium
KeywordsClostridium / Clostridioides / Binary / CDT / Iota / Toxin / TRANSLOCASE
Function / homology
Function and homology information


protein homooligomerization / transferase activity / nucleotide binding / extracellular region / identical protein binding
Similarity search - Function
Binary exotoxin A, clostridial type / 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 / ADP ribosyltransferase ...Binary exotoxin A, clostridial type / 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 / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / 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: 3.8 Å
AuthorsSheedlo MJ / Anderson DM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI095755 United States
Other governmentBX002943 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structural elucidation of the transferase toxin reveals a single-site binding mode for the enzyme.
Authors: Michael J Sheedlo / David M Anderson / Audrey K Thomas / D Borden Lacy /
Abstract: is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of infection are caused by the activity of three large toxins known ... is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of infection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and the transferase toxin (CDT). Reported here is a 3.8-Å cryo-electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to β-barrel pore, although we also observe a single bound CDTa in the prepore and β-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.
History
DepositionNov 21, 2019-
Header (metadata) releaseMar 4, 2020-
Map releaseMar 18, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v1s
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21016.png

Downloads & links

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Map

FileDownload / File: emd_21016.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM Reconstruction of CDT
Voxel sizeX=Y=Z: 1.13 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.056753296 - 0.10183433
Average (Standard dev.)0.0002635911 (±0.004621967)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 248.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.131.131.13
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z248.600248.600248.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0570.1020.000

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Supplemental data

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Sample components

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Entire : C. difficile transferase toxin

EntireName: C. difficile transferase toxin
Components
  • Complex: C. difficile transferase toxin
    • Protein or peptide: ADP-ribosyltransferase binding componentPoly (ADP-ribose) polymerase
    • Protein or peptide: ADP-ribosylating binary toxin enzymatic subunit CdtA
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: C. difficile transferase toxin

SupramoleculeName: C. difficile transferase toxin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 600 KDa

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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: 98.916828 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIQMRNKKV LSFLTLTAIV SQALVYPVYA QTSTSNHSNK KKEIVNEDIL PNNGLMGYYF TDEHFKDLKL MAPIKDGNLK FEEKKVDKL LDKDKSDVKS IRWTGRIIPS KDGEYTLSTD RDDVLMQVNT ESTISNTLKV NMKKGKEYKV RIELQDKNLG S IDNLSSPN ...String:
MKIQMRNKKV LSFLTLTAIV SQALVYPVYA QTSTSNHSNK KKEIVNEDIL PNNGLMGYYF TDEHFKDLKL MAPIKDGNLK FEEKKVDKL LDKDKSDVKS IRWTGRIIPS KDGEYTLSTD RDDVLMQVNT ESTISNTLKV NMKKGKEYKV RIELQDKNLG S IDNLSSPN LYWELDGMKK IIPEENLFLR DYSNIEKDDP FIPNNNFFDP KLMSDWEDED LDTDNDNIPD SYERNGYTIK DL IAVKWED SFAEQGYKKY VSNYLESNTA GDPYTDYEKA SGSFDKAIKT EARDPLVAAY PIVGVGMEKL IISTNEHAST DQG KTVSRA TTNSKTESNT AGVSVNVGYQ NGFTANVTTN YSHTTDNSTA VQDSNGESWN TGLSINKGES AYINANVRYY NTGT APMYK VTPTTNLVLD GDTLSTIKAQ ENQIGNNLSP GDTYPKKGLS PLALNTMDQF SSRLIPINYD QLKKLDAGKQ IKLET TQVS GNFGTKNSSG QIVTEGNSWS DYISQIDSIS ASIILDTENE SYERRVTAKN LQDPEDKTPE LTIGEAIEKA FGATKK DGL LYFNDIPIDE SCVELIFDDN TANKIKDSLK TLSDKKIYNV KLERGMNILI KTPTYFTNFD DYNNYPSTWS NVNTTNQ DG LQGSANKLNG ETKIKIPMSE LKPYKRYVFS GYSKDPLTSN SIIVKIKAKE EKTDYLVPEQ GYTKFSYEFE TTEKDSSN I EITLIGSGTT YLDNLSITEL NSTPEILDEP EVKIPTDQEI MDAHKIYFAD LNFNPSTGNT YINGMYFAPT QTNKEALDY IQKYRVEATL QYSGFKDIGT KDKEMRNYLG DPNQPKTNYV NLRSYFTGGE NIMTYKKLRI YAITPDDREL LVLSVD

UniProtKB: ADP-ribosyltransferase binding component

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Macromolecule #2: ADP-ribosylating binary toxin enzymatic subunit CdtA

MacromoleculeName: ADP-ribosylating binary toxin enzymatic subunit CdtA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Glycosyltransferases; Pentosyltransferases
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 53.323336 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKFRKHKRI SNCISILLIL YLTLGGLLPN NIYAQDLQSY SEKVCNTTYK APIERPEDFL KDKEKAKEWE RKEAERIEQK LERSEKEAL ESYKKDSVEI SKYSQTRNYF YDYQIEANSR EKEYKELRNA ISKNKIDKPM YVYYFESPEK FAFNKVIRTE N QNEISLEK ...String:
MKKFRKHKRI SNCISILLIL YLTLGGLLPN NIYAQDLQSY SEKVCNTTYK APIERPEDFL KDKEKAKEWE RKEAERIEQK LERSEKEAL ESYKKDSVEI SKYSQTRNYF YDYQIEANSR EKEYKELRNA ISKNKIDKPM YVYYFESPEK FAFNKVIRTE N QNEISLEK FNEFKETIQN KLFKQDGFKD ISLYEPGKGD EKPTPLLMHL KLPRNTGMLP YTNTNNVSTL IEQGYSIKID KI VRIVIDG KHYIKAEASV VSSLDFKDDV SKGDSWGKAN YNDWSNKLTP NELADVNDYM RGGYTAINNY LISNGPVNNP NPE LDSKIT NIENALKREP IPTNLTVYRR SGPQEFGLTL TSPEYDFNKL ENIDAFKSKW EGQALSYPNF ISTSIGSVNM SAFA KRKIV LRITIPKGSP GAYLSAIPGY AGEYEVLLNH GSKFKINKID SYKDGTITKL IVDATLIP

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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 61.34 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31377

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6v1s:
Structure of the Clostridioides difficile transferase toxin

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