Cryo-EM structure of Type Ⅲ-A CRISPR effector complex
Dear EditorIn bacteria and archaea,Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute a crRNA-guided surveillance complex that defends against invading nucleic elements.1 The CRISPR-Cas system can be divided into six types (Types Ⅰ-Ⅵ),which differ in terms of their nucleic acid target,number and arrangement of cas genes,and the composition of a silencing complex.The Type Ⅲ CRISPR-Cas system can be further classified into two main subtypes:Type Ⅲ-A and Type Ⅲ-B.The CRISPR-Cas effector complex of the Type Ⅲ CRISPR-Cas system consists of five subunits (Csm1-5) in Type Ⅲ-A (Fig.1a) and six subunits (Cmr1-6) in Type Ⅲ-B,both binding a crRNA composed of a typical repeat-derived 8-nucleotide (nt) 5”-handle followed by a long variable RNA-guide region.In contrast to Type Ⅰ and Ⅱ effectors that target dsDNA,Type Ⅲ effectors are sequence-specific RNases that cleave target RNA and also target RNA-stimulated nonspecific DNases.2 To date,although atomic resolution structures of Type Ⅲ-B effector complexes have been obtained,3,4 only low resolution electron microscopy (EM) structures of Csm effector complexes from different species have been reported,including S.solfataricus Csm complex (SsCsm),s T.thermophilus Csm complex (TtCsm),6 and T.onnurineus complex (ToCsm).7 These structures reveal the overall architectural features of the Csm complex,however,the precise mechanisms of the Type Ⅲ-A complex assembly and action remain elusive because of the lack of the atomic resolution structure.Recently,Type Ⅲ effector complexes were demonstrated to synthesize a novel second messenger,cyclic oligonucleotide (tetra-adenylate/hexaadenylate,cOA,/cOA6),for activating the nonspecific RNA degradation activity of Csm6/Csx1.8,9 The molecule synthesized by Csm1 requires the binding of noncomplementary target RNA to the Csm effector complex,but the mechanism involved in this process is still unclear.Moreover,for the Type Ⅲ effector complex,it remains unclear how the 3”-flanking sequence of target RNA controls the ssDNA cleavage.10 To obtain a better understanding of the assembly and molecular mechanisms of the Type Ⅲ-A complex,we purified the Csm effector complex of T.onnurineus (ToCsm effector complex) expressed in E.coli,performed single particle Cryo-EM studies and solved its structure at a resolution of 3.35 (A) (Supplementary information,Figs.S1-S3 and Table S1).
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We thank Dr.Boling Zhu,Dr.Xiaojun Huang,Dr.Gang Ji and Dr.Zhenxi Guo for Cryo-EM data collection at the Center for Biological Imaging;Institute of Biophysics.We are grateful to the staffs from BL19U beamline at Shanghai Synchrotron Radiation Facility for their assistance during data collection.We acknowledge Dr.Zhensheng Xie and Dr.Lili Niu for Mass spectrometry analysis at Lab of Proteomics of Institute of Biophysics,Chinese AcadXDB08010301 and XDB29010000;the National Science Foundation of China31670750
2019-01-12（万方平台首次上网日期，不代表论文的发表时间）
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1195-1197
