Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog  Takuo Osawa, Hideko Inanaga, Chikara Sato, Tomoyuki Numata  Molecular Cell  Volume 58, Issue 3, Pages 418-430 (May 2015) DOI: 10.1016/j.molcel.2015.03.018 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Reconstitution of the Chimeric Cmr Complex (A) The arrangements of the cmr genes in P. furiosus and A. fulgidus. Sequence identities between the respective proteins of these two species are shown. (B) Reconstitution of the hybrid complex composed of PfCmr2dHD-Cmr3 and AfCmr4-Cmr5-Cmr6. (C) Gel filtration chromatogram of the reconstituted chimeric Cmr23456. (D) SDS-PAGE analysis of the gel filtration fractions from (C). The pooled fractions are indicated. MK represents molecular markers. (E) RNA cleavage activity of the reconstituted chimeric Cmr complex. The 39-mer Pf7.01-crRNA (red and black) and 37-mer target RNA (blue), which bears a 6 nt overhang at the 5′ end, were used. See also Figures S1 and S4 and Table S1. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Crystal Structure of the ChiCmrΔ1-ssDNA Complex (A) Schematic representation of the crRNA-ssDNA duplex in the crystal. The base pairs within the duplex observed in the crystal are depicted by lines. Disordered nucleotides are colored green. (B) Overall structure of ChiCmrΔ1 bound to ssDNA. The 5′ and 3′ ends of the crRNA are shown. The color codes of each molecule are indicated. (C) Schematic representation of the complex with the same color codes. See also Figures S2, S3, and S5. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Subunit Assemblies of the Cmr Proteins (A) Ribbon representations of Cmr3, Cmr4, and Cmr6, colored as follows: palm (gray), finger (cyan), thumb (red), and wrist (blue). The Cmr6 thumb, which is disordered in ChiCmrΔ1, is depicted by a dashed line. (B) Helical filament composed of Cmr3, Cmr4.1–Cmr4.3, and Cmr6. Each subunit is color coded as in Figure 2. The thumbs of Cmr3N and all Cmr4 subunits are highlighted in red. (C) Helical filament composed of Cmr2, Cmr5.1, Cmr5.2, and Cmr6. For clarity, the D1–D3 domains of Cmr2 and the regions other than the Cmr6 wrist are colored gray. See also Figure S6. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Mechanism for Specifying the Multiple Cleavage Sites with 6 nt Intervals (A) Ribbon representation of the crRNA-ssDNA duplex, showing the periodic nucleotide displacements of both strands, which form 5 bp segments between kinks. The displaced nucleotides in the crRNA and ssDNA are shown in blue and green, respectively. (B) Intercalation of the Cmr4 thumbs (red) into the duplex with 6 nt intervals. (C) Schematic representations of the 2′-deoxy-substituted RNAs at position(s) 14, 20, and 26 of the targets. The nucleotide lengths of the observable products from each substrate are indicated. (D) Target cleavage reactions with the 2′-deoxy-substituted RNAs as substrates, revealing the cleavage sites in the target RNAs. The dotted line indicates noncontiguous lanes. (E) The scissile bonds between nucleotides 14 and 15 (right), 20 and 21 (middle), and 26 and 27 (left) are sandwiched between Cmr4.3 and Cmr5.2, Cmr4.2 and Cmr5.1, and Cmr4.1 and the D4 domain of Cmr2, respectively. (F) Periodic arrangements of the crucial aspartate in Cmr4, in close proximity to the scissile bonds. The distances between the Asp31 side chains and the target sites are indicated. (G) RNA cleavage reaction by the mutant protein-containing Cmr complex. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Repetitive Interactions with the Guide-Target Duplex (A) The cluster A residues from Cmr4.2 bind the phosphate backbone of the crRNA in the second 5 bp segment (stereoview). The hydrophobic residues (Ala263, Val265, and Leu279) from the Cmr4.1 thumb stabilize the 5 bp segment at one end. Hydrogen bonds are indicated as dotted lines. The nucleotide numbers of the crRNA are indicated. The coloring scheme is the same as in Figure 2. (B) Stereoview of the interactions between the cluster B residues from Cmr4.3 and the crRNA backbone of the second 5 bp segment. Trp280 from the Cmr4.2 thumb locks the 5 bp segment at the other end. (C) Binding mode between Cmr6 and the third 5 bp segment (stereoview). (D) Stereoview of the interactions between the Cmr3N thumb and the first 5 bp segment. See also Figures S2, S7, and S8. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Mechanisms of the 5′ Tag Recognition and Target Cleavage (A) The 5′ tag of the crRNA is buried by Cmr3 and Cmr4.1, where the thumbs of Cmr3N and Cmr3C as well as the L-loop stabilize its curved, S-shaped structure. The coloring scheme is the same as in Figure 2, except for the L-loop and the thumbs of Cmr3N and Cmr3C, which are highlighted in gray, pink, and blue, respectively. (B) Stereoview of the interactions between Cmr3 and the first two nucleotides of the 5′ tag, revealing the importance of U2 in Cmr assembly. Hydrogen bonds are indicated as dotted lines. (C) Stabilization of the quadruple base stack by Cmr3 (stereoview). (D) RNA cleavage activity of the mutant crRNA-containing Cmr complex. (E) Schematic representation of the target RNA cleavage mechanism. The Cmr complex cleaves it at multiple sites with 6 nt intervals, beginning 5 nt downstream from the 5′ tag. The crRNA 5′ tag is shown in white. The crucial aspartate residues are also indicated. See also Figure S2. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Structural and Functional Similarities between the Cmr and Cascade Complexes (A) Overall structures of the ChiCmrΔ1-ssDNA (left) and E. coli Cascade-ssDNA (right) complexes. (B) Mechanism for defining the 6 nt length of the crRNA guide by using the thumbs of Cmr4 (left) and Cas7 (right), by which the complementary nucleotides of the guide-target duplex are displaced with 6 nt intervals. (C) Interactions showing the functional similarities of Cmr3 (left) and Cas5 (right), which both cap the helical filament, bind the 5′ tag of the crRNA, and stabilize the start position of the duplex. The nucleotide numbers of the 5′ tag are indicated. (D) The second helical filaments observed in ChiCmrΔ1 (left) and Cascade (right). See also Figure S6. Molecular Cell 2015 58, 418-430DOI: (10.1016/j.molcel.2015.03.018) Copyright © 2015 Elsevier Inc. Terms and Conditions