+Open data
-Basic information
Entry | Database: PDB / ID: 6v1s | |||||||||
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Title | Structure of the Clostridioides difficile transferase toxin | |||||||||
Components |
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Keywords | TRANSLOCASE / Clostridium / Clostridioides / Binary / CDT / Iota / Toxin | |||||||||
Function / homology | Function and homology information protein homooligomerization / transferase activity / nucleotide binding / extracellular region / identical protein binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Clostridioides difficile (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Sheedlo, M.J. / Anderson, D.M. / Thomas, A.K. / Lacy, D.B. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6v1s.cif.gz | 475.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6v1s.ent.gz | 341.6 KB | Display | PDB format |
PDBx/mmJSON format | 6v1s.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6v1s_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 6v1s_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6v1s_validation.xml.gz | 62.7 KB | Display | |
Data in CIF | 6v1s_validation.cif.gz | 94.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/v1/6v1s ftp://data.pdbj.org/pub/pdb/validation_reports/v1/6v1s | HTTPS FTP |
-Related structure data
Related structure data | 21016MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 98916.828 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: cdtB / Production host: Escherichia coli (E. coli) / References: UniProt: A8DS70 #2: Protein | | Mass: 53323.336 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: cdtA, E5N70_18065, SAMEA897066_00723 / Production host: Escherichia coli (E. coli) References: UniProt: Q9KH42, Transferases; Glycosyltransferases; Pentosyltransferases #3: Chemical | ChemComp-CA / Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: C. difficile transferase toxin / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.6 MDa / Experimental value: NO |
Source (natural) | Organism: Clostridioides difficile (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1 |
Vitrification | Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 61.34 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 |
-Processing
Software | Name: PHENIX / Version: 1.16_3549: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31377 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||
Refinement | Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 48.61 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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