|Entry||Database: PDB / ID: 7oxd|
|Title||Crystal structure of Scytonema hofmanni transposition protein TniQ|
|Keywords||DNA BINDING PROTEIN / transposition protein / zinc-finger protein|
|Biological species||Scytonema hofmannii (Cyanobacteria)|
|Method||X-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.3 Å|
|Authors||Querques, I. / Jinek, M.|
|Funding support|| Switzerland, 2items |
|Citation||Journal: Nature / Year: 2021|
Title: Target site selection and remodelling by type V CRISPR-transposon systems.
Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.
|Structure viewer||Molecule: |
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|#1: Protein|| |
Mass: 17630.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (Cyanobacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
|#3: Water|| ChemComp-HOH / ||Has ligand of interest||Y|
|Experiment||Method: X-RAY DIFFRACTION / Number of used crystals: 1|
|Crystal||Density Matthews: 2.56 Å3/Da / Density % sol: 51.88 %|
|Crystal grow||Temperature: 280 K / Method: vapor diffusion, hanging drop|
Details: 100 mM HEPES pH 7.5 20% PEG 6000 250 mM sodium chloride
|Diffraction||Mean temperature: 100 K / Serial crystal experiment: N|
|Diffraction source||Source: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å|
|Detector||Type: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Aug 16, 2019|
|Radiation||Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray|
|Radiation wavelength||Wavelength: 1 Å / Relative weight: 1|
|Reflection||Resolution: 1.3→43.24 Å / Num. obs: 85839 / % possible obs: 99.75 % / Redundancy: 6.7 % / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.04553 / Rpim(I) all: 0.01314 / Rrim(I) all: 0.04743 / Net I/σ(I): 32.11|
|Reflection shell||Resolution: 1.3→1.346 Å / Redundancy: 12.1 % / Rmerge(I) obs: 0.9636 / Num. unique obs: 53615 / CC1/2: 0.889 / Rpim(I) all: 0.2847 / % possible all: 99.8|
|Refinement||Method to determine structure: SAD / Resolution: 1.3→43.24 Å / SU ML: 0.13 / Cross valid method: THROUGHOUT / σ(F): 1.29 / Phase error: 21.24 / Stereochemistry target values: ML|
|Solvent computation||Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL|
|Displacement parameters||Biso max: 50 Å2 / Biso mean: 20.7887 Å2 / Biso min: 10.96 Å2|
|Refinement step||Cycle: final / Resolution: 1.3→43.24 Å|
|LS refinement shell|
Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30
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