Computational protocol: Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases

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Protocol publication

[…] All diffraction data were indexed, integrated and scaled using XDS (). For TtCas6B, experimental phases were obtained from a three-wavelength multi-wavelength anomalous diffraction dataset collected at the Zn K-edge. The positions of zinc sites were determined using Phenix.hyss (). Phases were subsequently calculated and improved by density modification using solvent flipping using AutoSharp (). The resulting electron density maps were of excellent quality, and an initial model of TtCas6B could be readily built in COOT (). Iterative cycles of model building in COOT and refinement in Phenix.refine () yielded a final model with Rwork of 21.1% and Rfree of 23.9%. The structure of TtCas6B bound to R3 RNA was determined by molecular replacement using the Phaser module in Phenix (,). The apo-TtCas6B structure was used as the search model, and the resulting phases gave electron density maps that revealed the presence of two RNA molecules bound to the TtCas6B dimer. These were initially built in COOT using idealized dsRNA. The structure was completed by iterative building and refinement cycles using COOT and Phenix.refine. Final refinement statistics for all models are shown in Supplementary Table S1.All TtCas6A structures were solved by molecular replacement. Initially, the apo-TtCas6B atomic model was used to generate a search model for TtCas6A in Phenix. This model was subsequently used to solve the structure of the TtCas6A:R1 substrate RNA complex. Initial molecular replacement phases were improved using the prime-and-switch algorithm in RESOLVE () to produce a readily interpretable electron density map revealing two TtCas6A molecules and one RNA molecule in the asymmetric unit. The initial atomic model of TtCas6A was built automatically in Arp/wArp (), while the RNA molecule was built in COOT starting from an idealized RNA hairpin structure. The TtCas6A H37A and TtCas6A–R1 product complex structures were solved by molecular replacement using the refined model of TtCas6A and the R1 RNA from the substrate complex. Atomic models were built using COOT and refined in Phenix.refine. All models have excellent stereochemistry, as judged using the Molprobity server (), with more than 95% of all protein amino acid residues in the preferred regions of the Ramachandran plot and no Ramachandran outliers (Supplementary Table S1). […]

Pipeline specifications

Software tools XDS, PHENIX, Coot, ARP/wARP, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Thermus thermophilus