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Open data
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Basic information
Entry | Database: PDB / ID: 6pif | ||||||
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Title | V. cholerae TniQ-Cascade complex, open conformation | ||||||
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![]() | RNA BINDING PROTEIN/RNA / CRISPR/Cas / Cascade / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex | ||||||
Function / homology | RNA / RNA (> 10)![]() | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||
![]() | Halpin-Healy, T. / Klompe, S. / Sternberg, S.H. | ||||||
![]() | ![]() 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. | ||||||
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 705 KB | Display | ![]() |
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PDB format | ![]() | 570.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 20349MC ![]() 6pigC ![]() 6pijC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 3 types, 8 molecules ABCDEFGH
#1: Protein | Mass: 39517.586 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: Protein | | Mass: 58266.980 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #3: Protein | | Mass: 22999.234 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-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.) ![]() ![]() ![]() ![]() |
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#5: Protein | Mass: 42373.066 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-RNA chain , 1 types, 1 molecules 1
#6: RNA chain | Mass: 19237.338 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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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 |
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Sample preparation
Component | Name: VC-Tn667 transposon encoded CRISPR/Cas effector / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.6 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
Buffer solution | pH: 7.2 | ||||||||||||||||||||
Buffer component |
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Specimen | 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: UltrAuFoil | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
Software |
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EM software | Name: RELION / Version: 3 / Category: final Euler assignment | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85000 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3.4→3.4 Å / Cor.coef. Fo:Fc: 0.852 / SU B: 26.769 / SU ML: 0.385 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.819 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 142.69 Å2 / Biso mean: 89.648 Å2 / Biso min: 30 Å2
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Refinement step | Cycle: final / Resolution: 3.6→182.4 Å
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Refine LS restraints |
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LS refinement shell | Resolution: 3.6→3.693 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
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