+Open data
-Basic information
Entry | Database: PDB / ID: 7svw | ||||||
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Title | Strand-transfer complex of TnsB from ShCAST | ||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / CAST / transposase / strand-transfer complex / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex | ||||||
Function / homology | DNA / DNA (> 10) Function and homology information | ||||||
Biological species | [Scytonema hofmanni] UTEX 2349 (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.69 Å | ||||||
Authors | Park, J. / Tsai, A.W.T. / Kellogg, E.H. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM. Authors: Jung-Un Park / Amy Wei-Lun Tsai / Tiffany H Chen / Joseph E Peters / Elizabeth H Kellogg / Abstract: CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the ...CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7svw.cif.gz | 362.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7svw.ent.gz | 275.3 KB | Display | PDB format |
PDBx/mmJSON format | 7svw.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7svw_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7svw_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 7svw_validation.xml.gz | 61.6 KB | Display | |
Data in CIF | 7svw_validation.cif.gz | 91.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sv/7svw ftp://data.pdbj.org/pub/pdb/validation_reports/sv/7svw | HTTPS FTP |
-Related structure data
Related structure data | 25455MC 7svvC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: DNA chain | Mass: 21579.830 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #2: DNA chain | Mass: 13905.951 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #3: DNA chain | Mass: 6062.945 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #4: Protein | Mass: 66637.070 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) [Scytonema hofmanni] UTEX 2349 (bacteria) Production host: Escherichia coli (E. coli) #5: Chemical | Has ligand of interest | Y | |
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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: strand-transfer complex of TnsB from ShCAST element / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.35 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||||||||||||
Source (natural) | Organism: [Scytonema hofmanni] UTEX 2349 (bacteria) | ||||||||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 | ||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 49 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||
Particle selection | Num. of particles selected: 186121 | |||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | |||||||||||||||
3D reconstruction | Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46568 / Algorithm: BACK PROJECTION / Symmetry type: POINT | |||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL |