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- EMDB-32043: Complex structure of Clostridioides difficile enzymatic component... -

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Basic information

Entry
Database: EMDB / ID: EMD-32043
TitleComplex structure of Clostridioides difficile enzymatic component (CDTa) and binding component (CDTb) pore with long stem
Map data
Sample
  • Complex: CDTa-bound CDTb-pore (long)
    • Complex: CDTb-pore
      • Protein or peptide: ADP-ribosylating binary toxin binding subunit CdtB
    • Complex: CdtA
      • Protein or peptide: CdtA
  • Ligand: CALCIUM ION
KeywordsComplex / Translocation / Oligomer / Unfoldase / TOXIN
Function / homology
Function and homology information


protein homooligomerization / transferase activity / extracellular region / metal ion binding / 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 / CdtA
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsYamada T / Kawamoto A / Yoshida T / Sato Y / Kato T / Tsuge H
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
Japan Science and Technology Japan
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile.
Authors: Akihiro Kawamoto / Tomohito Yamada / Toru Yoshida / Yusui Sato / Takayuki Kato / Hideaki Tsuge /
Abstract: Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically ...Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically important bacterium with such a system is Clostridioides difficile (formerly Clostridium). The CDTa and CDTb proteins from its system represent important therapeutic targets. CDTb has been proposed to be a di-heptamer, but its physiological heptameric structure has not yet been reported. Here, we report the cryo-EM structure of CDTa bound to the CDTb-pore, which reveals that CDTa binding induces partial unfolding and tilting of the first CDTa α-helix. In the CDTb-pore, an NSS-loop exists in 'in' and 'out' conformations, suggesting its involvement in substrate translocation. Finally, 3D variability analysis revealed CDTa movements from a folded to an unfolded state. These dynamic structural information provide insights into drug design against hypervirulent C. difficile strains.
History
DepositionOct 11, 2021-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32043.png

Downloads & links

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Map

FileDownload / File: emd_32043.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 432 pix.
= 380.16 Å		0.88 Å/pix.
x 432 pix.
= 380.16 Å		0.88 Å/pix.
x 432 pix.
= 380.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0154
Minimum - Maximum-0.058233526 - 0.11971574
Average (Standard dev.)0.0001739695 (±0.0025936977)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 380.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_32043_additional_1.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 : CDTa-bound CDTb-pore (long)

EntireName: CDTa-bound CDTb-pore (long)
Components
  • Complex: CDTa-bound CDTb-pore (long)
    • Complex: CDTb-pore
      • Protein or peptide: ADP-ribosylating binary toxin binding subunit CdtB
    • Complex: CdtA
      • Protein or peptide: CdtA
  • Ligand: CALCIUM ION

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Supramolecule #1: CDTa-bound CDTb-pore (long)

SupramoleculeName: CDTa-bound CDTb-pore (long) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Clostridioides difficile (bacteria)

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Supramolecule #2: CDTb-pore

SupramoleculeName: CDTb-pore / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Clostridioides difficile (bacteria)

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Supramolecule #4: CdtA

SupramoleculeName: CdtA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: ADP-ribosylating binary toxin binding subunit CdtB

MacromoleculeName: ADP-ribosylating binary toxin binding subunit CdtB / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 75.657141 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: NNNFFDPKLM SDWEDEDLDT DNDNIPDSYE RNGYTIKDLI AVKWEDSFAE QGYKKYVSNY LESNTAGDPY TDYEKASGSF DKAIKTEAR DPLVAAYPIV GVGMEKLIIS TNEHASTDQG KTVSRATTNS KTESNTAGVS VNVGYQNGFT ANVTTNYSHT T DNSTAVQD ...String:
NNNFFDPKLM SDWEDEDLDT DNDNIPDSYE RNGYTIKDLI AVKWEDSFAE QGYKKYVSNY LESNTAGDPY TDYEKASGSF DKAIKTEAR DPLVAAYPIV GVGMEKLIIS TNEHASTDQG KTVSRATTNS KTESNTAGVS VNVGYQNGFT ANVTTNYSHT T DNSTAVQD SNGESWNTGL SINKGESAYI NANVRYYNTG TAPMYKVTPT TNLVLDGDTL STIKAQENQI GNNLSPGDTY PK KGLSPLA LNTMDQFSSR LIPINYDQLK KLDAGKQIKL ETTQVSGNFG TKNSSGQIVT EGNSWSDYIS QIDSISASII LDT ENESYE RRVTAKNLQD PEDKTPELTI GEAIEKAFGA TKKDGLLYFN DIPIDESCVE LIFDDNTANK IKDSLKTLSD KKIY NVKLE RGMNILIKTP TYFTNFDDYN NYPSTWSNVN TTNQDGLQGS ANKLNGETKI KIPMSELKPY KRYVFSGYSK DPLTS NSII VKIKAKEEKT DYLVPEQGYT KFSYEFETTE KDSSNIEITL IGSGTTYLDN LSITELNSTP EILDEPEVKI PTDQEI MDA HKIYFADLNF NPSTGNTYIN GMYFAPTQTN KEALDYIQKY RVEATLQYSG FKDIGTKDKE MRNYLGDPNQ PKTNYVN LR SYFTGGENIM TYKKLRIYAI TPDDRELLVL SVD

UniProtKB: ADP-ribosyltransferase binding component

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Macromolecule #2: CdtA

MacromoleculeName: CdtA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 49.420469 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: APIERPEDFL KDKEKAKEWE RKEAERIEQK LERSEKEALE SYKKDSVEIS KYSQTRNYFY DYQIEANSRE KEYKELRNAI SKNKIDKPM YVYYFESPEK FAFNKVIRTE NQNEISLEKF NEFKETIQNK LFKQDGFKDI SLYEPGKGDE KPTPLLMHLK L PRNTGMLP ...String:
APIERPEDFL KDKEKAKEWE RKEAERIEQK LERSEKEALE SYKKDSVEIS KYSQTRNYFY DYQIEANSRE KEYKELRNAI SKNKIDKPM YVYYFESPEK FAFNKVIRTE NQNEISLEKF NEFKETIQNK LFKQDGFKDI SLYEPGKGDE KPTPLLMHLK L PRNTGMLP YTNTNNVSTL IEQGYSIKID KIVRIVIDGK HYIKAEASVV SSLDFKDDVS KGDSWGKANY NDWSNKLTPN EL ADVNDYM RGGYTAINNY LISNGPVNNP NPELDSKITN IENALKREPI PTNLTVYRRS GPQEFGLTLT SPEYDFNKLE NID AFKSKW EGQALSYPNF ISTSIGSVNM SAFAKRKIVL RITIPKGSPG AYLSAIPGYA GEYEVLLNHG SKFKINKIDS YKDG TITKL IVDATLIPEN LYFQGLEHHH HHH

UniProtKB: CdtA

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 21 / 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

Concentration1.58 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4S2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
1.0 mMCaCl2Calcium chloride
0.003 % (w/v)C47H88O22Lauryl Maltose Neopentyl Glycol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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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: 11284 / Average exposure time: 3.36 sec. / Average electron dose: 50.0 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: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 735102
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 83061
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6uwr

source_name: PDB, initial_model_type: experimental model

6v1s

source_name: PDB, initial_model_type: experimental model
Output model

PDB-7vnn:
Complex structure of Clostridioides difficile enzymatic component (CDTa) and binding component (CDTb) pore with long stem

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