Crystallization and preliminary X‐ray diffraction analysis of the CRISPR–Cas RNA‐silencing Cmr complex. Issue 6 (1st June 2015)

• Record Type: Journal Article
• Authors: Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki
• Is Part Of: Acta crystallographica. Volume 71:Issue 6(2015:Jun.)
• Page Start: 735
• Page End: 740
• Publication Date: 2015-06-01
• Subjects: Crystallography -- Periodicals; Crystals -- Periodicals; 548
• DOI: 10.1107/S2053230X15007104
• Languages: English
• ISSNs: 2053-230X

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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 <italic>Pyrococcus furiosus</italic> Cmr2–Cmr3, <italic>Archaeoglobus fulgidus</italic> Cmr4–Cmr5–Cmr6 and the 39‐mer <italic>P. furiosus</italic> 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 <italic>P</italic>1, with unit‐cell parameters <italic>a</italic> = 75.5, <italic>b</italic> = 76.2, <italic>c</italic> = 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 ų Da⁻¹ and a solvent content of 39.5%.</p> </abstract>