Purification, crystallization and X-ray crystallographic analysis of Csm5 in Type III-A Crispr-Cas system

Abstract

The CRISPR-Cas system is a microbial adaptive and heritable immune system, of which mechanism relies on the effector ribonucleoprotein (RNP) complex to degrade foreign genetic elements. The effector surveillance complex of the Type III-A CRISPR-Cas system has five Csm components, Csm1~Csm5. The Csm5 protein is placed in the crRNA 3' ends in the effector RNP complex, leading to speculations that crRNA maturation may be catalyzed by the Csm5 subunit. However, the crystal structure and the detailed function of Csm5 still remain elusive. In this study, the Csm5 from Thermococcus onnurineus NA1 was purified and crystallized by the sitting drop method in the condition of 20% w/v PEG 8000 and 100 mM CHES/sodium hydroxide pH 9.5 at 291K. The diffraction data were collected to a resolution of 3.5 A. The crystal belonged to space group P4 3 2 1 2, with unit cell parameters a=81.0 A, b=81.0 A, c=169.1 A. One protomer was presented in the asymmetric unit with a corresponding V M of 2.10 A 3 Da -1 and solvent content of 50%.