Purification, crystallization and X-ray crystallographic analysis of Csm1/Csm4 subcomplex in CRISPR/Cas Type III-A system

Abstract

The CRISPR/Cas system is an adaptive prokaryotic immune response that defends against exogenous genetic elements. The CRISPR/Cas Type III-A system targets RNA and DNA that is coupled with transcription. The effector complex of the Type III-A CRISPR-Cas system has five Csm components, Csm1~Csm5. The Csm1/Csm4 sub-complex is placed in the crRNA 5' ends of the effector RNP complex and likely involved in the discriminative function for self vs non-self DNA. Here, we prepared Csm1/Csm4 proteins from Thermococcus onnurineus NA1 and crystallized the sub-complex. The Csm1/Csm4 was crystallized using hanging-drop vapor diffusion from a reservoir solution containg 200 mM sodium chloride, 100 mM sodium acetate trihydrate, pH 4.6, 26% (+/-)-2-methyl-2,4-pentanediol. The diffraction data to 2.8 A resolution show that the crystal belongs to the space group P6522 with unit cell parameters of a=154.91A, b=154.91A, c=182.25A, α=β=90° and γ=120°.