PMC

Projections of the first and second principal motions (PC1 vs PC2), derived from MD simulations of the apo Cas9 (a), Cas9:RNA (b), Cas9:RNA:DNA (c), and Cas9:pre-cat (d) systems, characterizing the conformational space sampled by Cas9 into regions in which the protein is “open” (red cloud) and “closed” (blue cloud, Movies S1, S2, S3, and S4).

“Essential dynamics” (i.e., first principal component, PC1) of the HNH domain plotted on the protein molecular surface of the Cas9:RNA:DNA (a) and Cas9:pre-cat (b) systems. The RNA (orange), target DNA (t-DNA, blue), and non-target DNA (nt-DNA, black) strands are shown as tubes. In Cas9:pre-cat, the HNH domain moves toward the t-DNA, approaching the catalytic site (indicated using a red cloud) to the cleavage site, in contrast to Cas9:RNA:DNA.

“Essential dynamics”, derived from the first principal component (PC1), of the individual protein domains of the apo Cas9 (a), Cas9:RNA (b), Cas9:RNA:DNA (c), and Cas9:pre-cat (d) systems, shown using arrows of sizes proportional to the amplitude of motions. The RNA (orange), target DNA (t-DNA, blue), and non-target DNA (nt-DNA, black) strands are shown as tubes. For the sake of clarity, the largest amplitude motions are shown, with the Cas9 individual domains color-coded as in .

Two-by-two matrices of the accumulated per-residue correlation scores (Cs_i, reported as a normalized frequency), calculated for each protein domain of the four simulated systems. This identifies interdependent domain motions, occurring in lockstep (blue) or in opposite direction (red) with respect to each other (full details in the Supporting Information). The protein sequence is shown along the axes, highlighting individual protein domains with different colors.

Representative snapshots of Cas9:pre-cat without the non-target DNA (w/o nt-DNA) (a) and with the nt-DNA (b), from MD simulations. In the absence of the nt-DNA, the catalytic H840 moves to a distance of ∼25 Å from the scissile phosphate P-3 on the target DNA (t-DNA). In the presence of the nt-DNA, H840 approaches P-3 at ∼15 Å distance. Concurrently, the K913 residue in the L2 loop forms H-bonds with C-3 in the nt-DNA. The protein is shown in molecular surface, highlighting the HNH domain (green) and the L2 loop (blue, right panel) as cartoon. The t-DNA (blue) and nt-DNA (black) strands are shown as ribbons. The RNA is omitted for the sake of clarity. Key protein residues (e.g., H840 and K913) are shown as sticks. The bottom graph reports time evolution of the distance between H840 (Cα atom) and the scissile phosphate P-3, during MD simulations of Cas9:pre-cat with nt-DNA (top) and without nt-DNA (bottom), color-coded according to the scale on the right.

X-ray structures of the apo Cas9 (4CMQ) (a), in complex with RNA (Cas9:RNA, 4ZT0) (b), and in the DNA-bound states (c, d), as captured in the presence of an incomplete DNA (Cas9:RNA:DNA, 4UN3) (c), and in a precatalytic state (Cas9:pre-cat, 5F9R) including both unwound strands (d). Cas9 is shown as cartoons, highlighting individual protein domains with different colors. The RNA (orange), target DNA (t-DNA, blue), and non-target DNA (nt-DNA, black) strands are in ribbons. In the apo Cas9 and Cas9:RNA structures, the α-helical lobe regions RECI (silver), RECII (gray), and RECIII (black) adopt remarkably different configurations; whereas different conformations of the HNH domain (green) are observed in the DNA-bound states. In Cas9:RNA:DNA, the HNH catalytic site (red) points in the opposite direction with respect to the cleavage site in the t-DNA, while in Cas9:pre-cat, it repositions itself toward the t-DNA, although remaining at a ∼18 Å distance from the cleavage site.