1. Volume 56, Issue 1, Pages 43-54 (October 2014)
Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4 
Christian Benda, Judith Ebert, Richard A. Scheltema, Herbert B. Schiller, Marc Baumgärtner, Fabien Bonneau, Matthias Mann, Elena Conti 
Molecular Cell 
Volume 56, Issue 1, Pages (October 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 43-54DOI: (10.1016/j.molcel.2014.09.002)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
The HD Nuclease Domain of Pf Cmr2 from the Structure of the Full-Length Protein
(A) Crystal structure of Pf Cmr2 shown in a cartoon representation in two orthogonal orientations with the HD domain in violet and the adenylate cyclase (D1 and D3), Thumb (D2), and Cmr5-like (D4) domains in blue. The manganese and zinc ions are in gray and yellow, respectively. The left panel highlights the secondary structure elements of the HD domain.
(B) Superposition of full-length Cmr2 with a published Cmr2-Cmr3 structure (PDB ID 3W2V, Cmr3 in green) shows the overall shape of the Cmr2-Cmr3 building block used in fitting of the Cmr complex.
(C) The zoom highlights the active-site residues of the HD domain.
Molecular Cell  , 43-54DOI: ( /j.molcel )
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4. Figure 2
Features of the Cas7-like Proteins from the Structures of Pf Cmr1, Cmr4, and Cmr6
(A) Topology diagram of the RRM domain of the Cas7 protein family as referred to in the text. Helices are represented as circles and β strands as arrows, and they are labeled numerically. The position of insertion domains characteristic of the Cas7 protein family is indicated.
(B) Crystal structure of Pf Cmr1 (yellow), with the N- and C-terminal RRM domains indicated. The lid domain of the N-terminal RRM is indicated. The zoom on the right shows a ribonucleotide bound at the interface of the two RRM (with the 2Fo-Fc density contoured at 1.5σ).
(C and D) Crystal structures of Pf Cmr4 (red) and Cmr5 (pink) with structural features indicated. On the right are the surface representations of the superhelical filaments in the lattices, with the molecules in the asymmetric unit colored in light and dark shades.
(E) Crystal structure of Pf Cmr6 (orange) with structural features indicated.
Molecular Cell  , 43-54DOI: ( /j.molcel )
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5. Figure 3 CXMS Identification of Lysine-Lysine Crosslinks
(A) Extracted ion chromatograms for a representative interprotein crosslink with average identification score. The low-energy CXD scan enables selective cleavage of the chemical crosslinker. This scan is continuously alternated with a survey scan where the crosslinked peptide pair remains intact (FULL) (cycle time ∼1.8 s). The relative changes in intensity of the crosslinked peptides (top) and the two fragmentation products (bottom two panels) in these alternate full scans are shown over time.
(B) Two consecutive scans in either the FULL scan (top), in which only the crosslinked peptide pair (black arrow) is identified, or the CXD scan (bottom), in which the two separated peptides appear (red and purple arrows).
(C) Peptides are selected from the CXD scan to be identified by high collision energy dissociation of the peptides, resulting in y-ion (red) and b-ion (blue) series fragments. The acquired MS/MS spectrum for the Cmr2 peptide is shown in the left panel, and that for the Cmr4 peptide is shown in the right panel.
(D) Schematic representation of all identified crosslinks. Interlinks between the Cmr complex subunits are depicted as black lines (example crosslink shown in A–C is highlighted in red). Intralinks and looplinks are depicted as gray lines and monolinks as gray dots. For interprotein links, every lysine has the color of the Cmr subunit it is connected to, and the size of the circle is scaled according to the lowest peptide identification score of the peptide pair.
Molecular Cell  , 43-54DOI: ( /j.molcel )
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6. Figure 4 Pseudoatomic Model of the Cmr Complex
(A) View of two orientations of the resolution cryo-EM map of the Pf Cmr complex (EMD-5740) fitted with the atomic models of the individual Cmr proteins. Molecules are color coded as in Figure 1. The small, unaccounted density is near the N terminus of Cmr6ΔN.
(B) Cmr complex topology represented by interprotein crosslinks (Cα-Cα distances < 35 Å) and simplified representations of the Cmr building blocks. For visibility, the model is shown in an exploded view. Multiple crosslinks between Cmr2 (D2 and D4) and Cmr4 or Cmr5 demonstrate the close interaction and relative orientation of these subunits. Crosslinks from Cmr3 to Cmr4 outline a binding epitope for Cmr4. Interprotein links between identical proteins (Cmr4-Cmr4 and Cmr5-Cmr5) are shown as dashed lines. Only one crosslink was found between Cmr4 and Cmr5, Cmr6 and Cmr4, and Cmr6 and Cmr5. Cmr1 was omitted in the CXMS experiment.
(C–F) Zoomed-in views showing selected crosslinks used to build and validate a pseudoatomic model of the Cmr complex. Lysine Cα atoms are shown as spheres and crosslinks as black lines. (C) Interlinks between adjacent Cmr4 copies; (D) links between Cmr4 and D4 of Cmr2; (E) interlink in the Cmr5 filament; (F) links between the structurally homologous Cmr5 and D4 of Cmr2.
Molecular Cell  , 43-54DOI: ( /j.molcel )
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7. Figure 5 The Active Site of Cmr4
(A) Left: final model of the Pf Cmr complex looking onto the central cleft between Cmr4 (red) and Cmr5 (pink). Subunits are color coded. To the right is an illustration showing a 45 nt crRNA and the resulting multiple cleavage sites on a target RNA, 14 nucleotides upstream of the 3′ end of the crRNA.
(B) Top: zoom on the central cleft with Cmr4 now colored according to sequence conservation (red, highly conserved; white, not conserved). Locations of putative active site residues in Cmr4 subunit D are indicated. Bottom: detailed view on the active site of Cmr4 with the residues tested shown as sticks and color coded according to conservation.
(C) Top: target cleavage assays. Cmr proteins mixed to obtain a 1 μM final complex concentration were incubated 2 hr at 55°C with 20 nM 45 nt psi-RNA guide and 20 nM 5′-32P-labeled 7.01 target RNA. Wild-type (WT) or indicated Cmr4 mutants, as well as Cmr5, were added to a 3-fold molar excess compared to the other subunits. Bottom: SDS-PAGE of proteins used to reconstitute the complex. A total of 20 pmol of the indicated proteins and complex were separated by 12% SDS-PAGE and stained with Coomassie blue. The asterisk indicates a contaminant present in the Cmr2 preparation.
(D) Top: sequence alignment of Cmr4 orthologs from P. furiosus (Pf), T. maritima (Tm), T. thermophilus (Tt), and S. solfataricus (Sso). Conserved residues are in colored boxes. The position of the nucleolytically active D26 is indicated, as well as other residues tested in the mutational screen. Bottom: alignment of Cmr4 against Cms3 orthologs, the putative backbone Cas7-protein from type III-A systems. Aspartate D26 is also conserved in these proteins (Methanopyrus kandleri [Mk], P. horikoshii [Ph], Sulfolobus islandicus [Si]). More comprehensive alignments are shown in Figure S5.
Molecular Cell  , 43-54DOI: ( /j.molcel )
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