title: Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.
abstract: Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566, Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Osawa
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566, Japan.
authors.forename: Hideko
authors.initials: H
authors.lastname: Inanaga
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Numata
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D019611:Archaeoglobus fulgidus / Q000737:chemistry* / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020027:Pyrococcus furiosus / Q000737:chemistry* / Q000378:metabolism; D034622:RNA Interference; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D000094704:RNA, Guide, CRISPR-Cas Systems; D011994:Recombinant Proteins
keywords: CRISPR-Cas; Cmr complex; crRNA