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- PDB-6klw: Complex structure of Iota toxin enzymatic component (Ia) and bind... -

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Basic information

Entry
Database: PDB / ID: 6klw
TitleComplex structure of Iota toxin enzymatic component (Ia) and binding component (Ib) pore with long stem
Components
  • Iota toxin component Ia
  • Iota toxin component Ib
KeywordsTOXIN / Bacterial binary toxin / Protein translocation channel / ADP-ribosylation
Function / homology
Function and homology information


protein homooligomerization / nucleotide binding / extracellular region
Similarity search - Function
Ubiquitin-like (UB roll) - #110 / 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 ...Ubiquitin-like (UB roll) - #110 / 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 / Toxin ADP-ribosyltransferase; Chain A, domain 1 / Toxin ADP-ribosyltransferase; Chain A, domain 1 / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / PA14 / PA14 domain / Ubiquitin-like (UB roll) / Roll / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Iota toxin component Ia / Iota toxin component Ib
Similarity search - Component
Biological speciesClostridium perfringens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsYoshida, T. / Yamada, T. / Kawamoto, A. / Mitsuoka, K. / Iwasaki, K. / Tsuge, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science18K06170 Japan
Japan Society for the Promotion of Science17K15095 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex.
Authors: Tomohito Yamada / Toru Yoshida / Akihiro Kawamoto / Kaoru Mitsuoka / Kenji Iwasaki / Hideaki Tsuge /
Abstract: The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and ...The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.
History
DepositionJul 30, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 27, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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Assembly

Deposited unit
A: Iota toxin component Ib
B: Iota toxin component Ib
C: Iota toxin component Ib
D: Iota toxin component Ib
E: Iota toxin component Ib
F: Iota toxin component Ib
G: Iota toxin component Ib
H: Iota toxin component Ia
hetero molecules


Theoretical massNumber of molelcules
Total (without water)569,27522
Polymers568,7148
Non-polymers56114
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47660 Å2
ΔGint-71 kcal/mol
Surface area138140 Å2

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Components

#1: Protein
Iota toxin component Ib


Mass: 74378.820 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Plasmid: pGEX-4T-1 / Production host: Escherichia coli (E. coli) / Strain (production host): Origami / References: UniProt: Q46221
#2: Protein Iota toxin component Ia


Mass: 48061.988 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Plasmid: pET15b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q46220
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex structure of Iota toxin enzymatic component (Ia) and binding component (Ib) pore with long stem
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.57 MDa / Experimental value: NO
Source (natural)Organism: Clostridium perfringens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
21 mMcalcium chlorideCaCl21
30.003 %LMNG1
SpecimenConc.: 0.48 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 78.9 K / Temperature (min): 78.9 K
Image recordingAverage exposure time: 84.09 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2151
EM imaging opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: (1.15.2_3472: phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimera1.14model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.15.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 871264
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62940 / Symmetry type: POINT
Atomic model buildingB value: 29 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3J9C

3j9c


Accession code: 3J9C / Source name: PDB / Type: experimental model

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