Computational protocol: Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

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Protocol publication

[…] The initial experimental phases were determined by single-wavelength anomalous dispersion (SAD) method using a data set collected at the tantalum L-III absorption edge from a crystal derivatized with Ta6Br12 cluster. The positions of 4 cluster sites were located using SHELXC/D. Low-resolution SAD phasing was carried out at 6.0 Å using Phenix.autosol, in which the four Ta6Br12 clusters were treated as superatoms. The resulting phases were then improved and extended to 3.0A using Phenix.autobuild by applying the noncrystallography symmetry (NCS) identified by Molrep. The Ta cluster phases allowed the placement of some alpha-helices and beta-sheets into the electron density map. To reliably trace the entire Cas3 molecule, we switched to the 3.5 Å Se-SAD data set, where 51 different Se sites were identified from the anomalous difference electron density map by combining the Se-SAD anomalous diffraction data set with the Ta phases. These sites provided enough phasing power to generate a traceable electron density map and partial models for all four copies of Cas3 molecules using the automated experimental phasing and model building procedures in Phenix.autosol and Phenix.autobuild. The phases were further improved by density modification using DM, and the model was manually rebuilt into the electron density map using Coot. The result model was refined using REFMAC5 and Phenix.refine. The Rfree statistics of the final structure models are well below 30%, with good r.m.s. deviations in bond length and bond angle. The backbone conformations of ~93–96% of the protein residues are in the most favored region in the Ramachandran plot, ~4–7% in the allowed region, and none in the disallowed region. The geometry of coordinates was validated by MOLPROBITY. All figures were generated using PyMOL (http://www.pymol.org). […]

Pipeline specifications

Software tools SHELX, PHENIX, Molrep, Coot, REFMAC5, MolProbity, PyMOL
Application Protein structure analysis
Organisms Thermobifida fusca
Diseases Huntington Disease