[English] 日本語
Yorodumi- PDB-6pij: Target DNA-bound V. cholerae TniQ-Cascade complex, closed conformation -
+Open data
-Basic information
Entry | Database: PDB / ID: 6pij | ||||||
---|---|---|---|---|---|---|---|
Title | Target DNA-bound V. cholerae TniQ-Cascade complex, closed conformation | ||||||
Components |
| ||||||
Keywords | RNA BINDING PROTEIN/RNA/DNA / CRISPR/Cas / Cascade / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex | ||||||
Function / homology | DNA / DNA (> 10) / RNA / RNA (> 10) Function and homology information | ||||||
Biological species | Vibrio cholerae (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
Authors | Halpin-Healy, T. / Klompe, S. / Sternberg, S.H. | ||||||
Citation | Journal: Nature / Year: 2020 Title: Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system. Authors: Tyler S Halpin-Healy / Sanne E Klompe / Samuel H Sternberg / Israel S Fernández / Abstract: Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically ...Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications. | ||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6pij.cif.gz | 727.9 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb6pij.ent.gz | 589.6 KB | Display | PDB format |
PDBx/mmJSON format | 6pij.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6pij_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 6pij_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 6pij_validation.xml.gz | 102.7 KB | Display | |
Data in CIF | 6pij_validation.cif.gz | 159.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pi/6pij ftp://data.pdbj.org/pub/pdb/validation_reports/pi/6pij | HTTPS FTP |
-Related structure data
Related structure data | 20351MC 6pifC 6pigC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
-Protein , 3 types, 8 molecules ABCDEFGH
#1: Protein | Mass: 39588.664 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli) #2: Protein | | Mass: 57064.711 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli) #3: Protein | | Mass: 22813.025 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli) |
---|
-TniQ monomer ... , 2 types, 2 molecules IJ
#4: Protein | Mass: 41517.160 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli) |
---|---|
#5: Protein | Mass: 42315.031 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli) |
-RNA chain , 1 types, 1 molecules 1
#6: RNA chain | Mass: 19237.338 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria) |
---|
-DNA chain , 2 types, 2 molecules 23
#7: DNA chain | Mass: 8391.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria) |
---|---|
#8: DNA chain | Mass: 2651.750 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio cholerae (bacteria) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: VC-Tn667 transposon encoded CRISPR/Cas effector / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 0.6 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Vibrio cholerae (bacteria) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||
Buffer solution | pH: 7.2 | ||||||||||||||||||||
Buffer component |
| ||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Details: unspecified | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: REFMAC / Version: 5.8.0238 / Classification: refinement |
---|---|
EM software | Name: RELION / Version: 3 / Category: final Euler assignment |
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74366 / Symmetry type: POINT |