5xvo

E. fae Cas1-Cas2/prespacer/target ternary complex revealing DNA sampling and half-integration states

spinqualitylabelsall models 5xvo, resolution 3.10Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5xvo is a 18 chain structure with sequence from Enterococcus faecalis TX0027. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 3.1Å Ligands: MG Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1-5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6-9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical7-9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle6,10. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3'-OH of each 3'-overhang serves as an attacking nucleophile during integration11-14. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat7,15-20. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)21,22. In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro18; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis17,23. Focusing on the Enterococcus faecalis Type II-A system24, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3'-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3'-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference. How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.,Xiao Y, Ng S, Nam KH, Ke A Nature. 2017 Sep 4. doi: 10.1038/nature24020. PMID:28869593 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Endonuclease 3D structures

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