Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance Patrick Pausch, Hanna Müller-Esparza, Daniel Gleditzsch, Florian Altegoer, Lennart Randau, Gert Bange  Molecular Cell  Volume 67, Issue 4, Pages 622-632.e4 (August 2017) DOI: 10.1016/j.molcel.2017.06.036 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Crystal Structure of the Short Type I-Fv Cascade Cartoon representation of the short I-Fv Cascade X-ray crystal structure from S. putrefaciens CN32 in two, 90° rotated orientations. Short crRNA, Cas5fv, Cas7fv, and Cas6f are colored in orange, dark red, blue, and green, respectively. The crRNA 3′ hairpin and 5′ end are indicated, and the Cascade subunits are labeled. Disordered sections are labeled and indicated by thin dotted lines. The two parallel right-handed wrist and palm/thumb helices are labeled accordingly and are indicated by thick dotted lines. The scale bar illustrating the total height of 130 Å is shown on the right. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Structural Comparison of Type I-F and Type I-Fv Cascade (A) Left: X-ray crystal structure of the short S. putrefaciens I-Fv Cascade shown in a cartoon representation. The color scheme and labeling are as in Figure 1. crRNA spine superimposition of the short I-Fv (orange) and I-F (gray) crRNA on the basis of a 5′ handle alignment is shown in the middle. Nucleotide positions upstream of the first spacer nucleotide are labeled with negative values, and the positions of downstream nucleotides are indicated by positive values. The angle of 24° between nucleotide position −6 and 12 illustrates the different crRNA spine pitch. cryo-EM structure of the AcrF1/2-bound P. aeruginosa I-F Cascade (PDB ID: 5ZU9; Chowdhury et al., 2017) is shown on the right. The components are labeled according to the current nomenclature for type I-F. The color scheme of the I-F Cas homologs is according to I-Fv. The additionally present large subunit protein Cas8f is shown in yellow, and the activity inhibiting AcrF1/2 proteins are shown in a gray surface representation. (B and C) Side-by-side comparison of the I-Fv (B) and I-F (C) Cascade subunits. The color is according to Figure 2A. The Cas6f proteins are compared in the left panel, Cas7 homologs in the middle panel, and Cas5 homologs in the right panel. Adjacent Cascade subunits are shown as transparent surfaces and labeled, respectively. Grey circles indicate disordered regions. N and C indicate N- and C-termini, respectively. (D) crRNA spine comparison of I-F (orange) and I-Fv (gray). crRNA arrangement at the tilted head structure is shown on the left. The superimposition is according to the I-Fv nucleotides 6 to 12. crRNA architecture of the Cas7 backbone-bound segment is shown in the middle. The superimposition is according to the I-Fv nucleotides −1 to −4. At every sixth position, the nucleotide is splayed out from the base stacking segments (“kink”). The superimposition of the similar S-shaped 5′ handles is shown on the right. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Structural Cascade Variation in Response to AcrF1 and AcrF2 Side-by-side comparison of the I-Fv Cascade base structure and the homologous and AcrF1/2 vulnerable I-F Cascade base structure (PDB ID: 5ZU9; Chowdhury et al., 2017). Both structures are shown in cartoon representation and the Cascade body and head structures were removed for clarity. The Cas proteins were colored according to Figure 2. The anti-CRISPR proteins AcrF1.2 (gray) and AcrF2 (green) are highlighted by transparent surfaces. Regions to which AcrF1/2 associate in I-F Cascade (i.e., Cas7f thumb and web; Cas8f) differ drastically in their structure to I-Fv, not allowing binding of AcrF1/2 to the I-Fv Cascade. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 X-Ray Crystal Structure of the Short I-Fv Cascade R-Loop Complex (A) Overview of the R-loop Cascade crystal structure. Components are shown in cartoon representation and colored and labeled according to Figures 1 and 2. Nucleic acid components are highlighted for clarity by transparent surfaces (orange: short crRNA; red: DNA target strand; and violet: non-target strand). Important regions for nucleic acid interaction, detailed in (C) to (G), are tagged with numbers in white circles (1–4) for orientation. (B) Design of target and non-target primers for the reconstitution of the R-loop/I-Fv Cascade complex. The blue, red, and green lines indicate the interface between Cas7fv, Cas5fv, and Cas6f and the nucleic acids as observed in the R-loop/I-Fv Cascade structure. The arrows indicate amino acids of Cas5fv interacting with the GG-PAM. Gray letters indicate disordered nucleotides. (C–F) Close up view of the DNA interacting regions close to the Cas6f head structure (C), at the thumb of Cas7fv.3 (D), the base of the wrist helix (E), and the PAM recognition site in between the AH and SH of Cas5fv (F). Amino acid side chains in close proximity to nucleic acids are shown as sticks and are labeled according to their identity and position. Nucleic acids are labeled according to (B). (G) Detailed view on the GG-PAM, shown in stick representation. Adjacent nucleotides were removed for clarity. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Structural Reorganization of Type I-F Cascade upon R-Loop Formation (A) Superimposition of the short Apo Cascade (gray cartoon) and the R-loop-associated short Cascade (colored cartoon). The I-Fv Cascade undergoes structural rearrangements at the Cas6f head and Cas5fv AH domain, indicated by arrows and distances. (B) Superimposition along dsDNA segment of the aligned Apo (gray) and R-loop-bound (colored) short Cascades with an ideal B-form DNA (light blue). The PAM region is highlighted with a dashed line. (C) Close up on the superimposition with an ideal B-form DNA (Figure 4B) upstream of the GG-PAM, emphasizing steric clashes that would occur between the target strand DNA and α helix 3 (wedge helix) of the Cas5fv AH domain upon target and non-target strand association. Residues are labeled according to their identity and position. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Efficiency of Transformation of Cas5fv Mutants (A) Schematic representation of the EOT assay. BL21-AI E. coli cells carrying the wild-type or mutant variants (represented with stars) of the cas genes (in pRSFDuet-1) and a minimal CRISPR array (in pCDF-Duet-1) are transformed with a third target plasmid (pETDuet-1) and plated in the presence of the target plasmid resistance antibiotic. After over night incubation, the activity of the wild-type and mutant complexes is determined by calculating the EOT as a ratio of the colonies of the strain of interest versus the corresponding Cas HD domain mutant. (B) Exemplary EOT plates of the positive control (wild-type Cascade), its matching negative control (Cas3 HD mutant Cascade), and the Cas5fv ΔAH plates are shown after over night growth. (C) EOT calculation for the investigated mutants. Assays were performed in triplicate, and the error bars were calculated as SEM. (D) Stability of the ΔAH domain mutant. Recombinant Cascade variants (containing either His-Cas5fv or His-Cas5fv ΔAH) were purified via nickel-nitrilotriacetic acid (Ni-NTA)-affinity chromatography and gel filtration. Fractions of the dominant SEC peak were separated via SDS-PAGE and revealed stable Cascade complex formation. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 R-Loop Formation by Type I-E and I-Fv Cascade Side-by-side comparison of components and mechanism of target DNA recognition by type I-E and I-Fv Cascade as deduced from structural analyses. Mechanism of surveillance and R-loop formation by the E. coli type I-E Cascade (compare to Hayes et al., 2016) are shown in the upper panel. A glutamine wedge (arrow) of the large subunit Cse1 (Cas8e) opens the R-loop, and the non-target strand is guided by small subunits Cse2. The minimal S. putrefaciens type I-Fv Cascade structure revealed a shift of the AH domain upon target DNA binding, which results in wedge helix insertion (arrow), as shown in the lower panel. The non-target strand is guided along a trench route formed by Cas5fv and Cas7fv WLs. Molecular Cell 2017 67, 622-632.e4DOI: (10.1016/j.molcel.2017.06.036) Copyright © 2017 Elsevier Inc. Terms and Conditions