Structure of an RNA Silencing Complex of the CRISPR-Cas Immune System Michael Spilman, Alexis Cocozaki, Caryn Hale, Yaming Shao, Nancy Ramia, Rebeca Terns, Michael Terns, Hong Li, Scott Stagg  Molecular Cell  Volume 52, Issue 1, Pages 146-152 (October 2013) DOI: 10.1016/j.molcel.2013.09.008 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Overview of the P. furiosus CRISPR/Cas Locus and the Cmr Complex Structure Determined by Cryoelectron Microscopy (A) The genes encoding the Cmr complex protein subunits are color coded to match those used for structure images in subsequent figures. The CRISPR repeats are shown in black and spacers in various colors. (B) The mature 39 nt and 45 nt crRNAs contain a 5′ repeat-derived 8 nt sequence (5′ tag) and a 31 nt or 37 nt spacer-derived sequence (guide), respectively. The sequence of the target RNA used in RNA cleavage assays and assembly with the Cmr complex is shown in blue. (C) Color-coded EM density of the Cmr complex bound with the 45 nt crRNA and the target RNA is shown in two orientations. Cmr1, red; Cmr2, light blue; Cmr3, orange; Cmr4, three different shades of green; Cmr5, three different shades of yellow; and Cmr6, magenta. (D) Fitting of the crystal structure of Cmr2-Cmr3 complex to the foot of the Cmr complex in two orthogonal orientations. (E) Helical reconstruction of the Cmr4–Cmr5 filament structure. Circled regions indicate matched densities in both the Cmr complex and the Cmr4–Cmr5 filament. See also Figures S1–S3. Molecular Cell 2013 52, 146-152DOI: (10.1016/j.molcel.2013.09.008) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Assembly Process of the Cmr Complex (A) Left, a ribbon model of the assembled protein subunits of an intact target-bound Cmr complex. Right, capping at the foot of the Cmr complex by interaction of the homologous domain of Cmr2 with Cmr5 and of Cmr3 with Cmr4. Structural similarity between Cmr5 (Park et al., 2013; PDB ID 4GKF) and the D4 domain of Cmr2 is shown (root-mean-square deviation [rmsd] = 3.3 Å for 62 aligned Cα atoms). A homology model of Cmr4 constructed from Cas6 (sequence similarity 30%; Carte et al., 2008; PDB ID 3PKM) is also homologous to that of Cmr3 (rmsd = 3.4 Å for 136 aligned Cα atoms). (B) A micrograph of the Cmr4–Cmr5 filament and the class averages of Cmr4–Cmr5 filaments. (C) Model for assembly method of the Cmr complex. By acting as a nonextendable Cmr4–Cmr5 dimer, Cmr6 and Cmr2-Cmr3 complex cap the growth of the Cmr4–Cmr5 filament at the top and foot of the Cmr complex, respectively. See also Figures S2E and S4. Molecular Cell 2013 52, 146-152DOI: (10.1016/j.molcel.2013.09.008) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Positions of the crRNA and Target RNA within the Complex (A) RNA substrates used for crosslinking assays. The 45 nt crRNAs (Supplemental Experimental Procedures and Table S1, 1–3, 5–8) or tag RNAs (Supplemental Experimental Procedures and Table S1, 4) were generated by in vitro transcription and subsequent Cas6 digestion. Regions marked in yellow contain radiolabeled nucleotides. (B and C) Shown are labeled (i.e., crosslinked) (B) and Coomassie-stained (i.e., total) (C) proteins. The RNA from (A) used in each reaction is indicated beneath the lanes and correlates with (A). T1 and A denote RNases used in the experiment and visible in the Coomassie-stained gel (C). (D) Difference density to identify bound target RNA. The difference density was obtained by subtracting the negatively stained density of the Cmr complex in the absence of target RNA from that in the presence of target RNA. Left, the common density ± target RNA is shown in yellow and the difference density in blue (10σ). Right, the same view as that on the left but clipped to expose the difference density in the center of the complex. (E) Schematic structure of the holo Cmr complex showing the deduced path of the crRNA and target RNA. See also Table S1. Molecular Cell 2013 52, 146-152DOI: (10.1016/j.molcel.2013.09.008) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Conserved Organization of an RNA- and DNA-Targeting CRISPR-Cas Effector Complex The Cse and Cmr CRISPR-Cas systems include distantly related members of several broad categories of Cas proteins: large subunit (blue), small subunit (shades of yellow), Cas5 superfamily (orange), Cas7 superfamily (shades of green), and Cas6 superfamily proteins (gray). Our findings reveal a conserved functional organization of these classes of Cas proteins within the effector complexes. (A) Comparison of cryo-EM structures of E. coli Cascade (left) and P. furiosus Cmr complex (right). Segmented densities assigned to the subunits are color coded according to their broad classification. (B) Cartoon representations of the functional organization of the complexes. The Cas5 superfamily RAMP proteins (Cmr3 and Cas5e) are found near the 5′ tag of the crRNAs in both complexes. Large subunit proteins (Cmr2, member of the Cas10 superfamily, and Cse1, member of the Cas8 superfamily) are also found near the 5′ end of the crRNAs. Cas7 superfamily RAMP proteins (Cmr4, Cmr6, and Cmr1 in the Cmr complex and Cse4 in the Cse complex) and the small subunit of proteins (Cmr5 and Cse2) form the backbones of the helical structure that extends along the guide region of the crRNAs. In Cascade, the Cas6 superfamily RAMP protein (Cse3) remains associated with the CRISPR repeat sequence retained at the 3′ end of the crRNA. The Cse complex binds DNA targets that are then cleaved by Cas3. The Cmr complex cleaves complementary RNAs. Molecular Cell 2013 52, 146-152DOI: (10.1016/j.molcel.2013.09.008) Copyright © 2013 Elsevier Inc. Terms and Conditions