|Entry||Database: PDB / ID: 7pla|
|Title||Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA target|
|Keywords||DNA BINDING PROTEIN / Cas12k / sgRNA / target DNA / Protein-RNA-DNA complex / Tn7 / Transposition / CRISPR|
|Function / homology||DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)|
Function and homology information
|Biological species||Scytonema hofmannii (Cyanobacteria)|
synthetic construct (others)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å|
|Authors||Schmitz, M. / Jinek, M.|
|Funding support||European Union, 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: |
Downloads & links
C: DNA target strand
D: DNA non-target strand
|#1: Protein|| |
Mass: 73744.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (Cyanobacteria) / Production host: Escherichia coli (E. coli)
|#2: RNA chain|| |
Mass: 82376.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (Cyanobacteria)
|#3: DNA chain|| |
Mass: 15301.837 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
|#4: DNA chain|| |
Mass: 15497.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Ternary complex of ShCas12k with sgRNA and target dsDNA|
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
|Molecular weight||Value: 0.187 MDa / Experimental value: NO|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Specimen holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Electron dose: 51.81 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138000 / Symmetry type: POINT|
|Refinement||Cross valid method: NONE|
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
|Displacement parameters||Biso mean: 185.47 Å2|
|Refine LS restraints|
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