2D correlation coefficients were calculated between the HS-AFM images of the first frame and each of the frames within the Region of Interest (ROI) (i.e., the first frame is the reference)23. The sizes of the ROIs for apo-Cas9 and Cas9–RNA that enclosed the whole Cas9 molecule were about 27 × 24 nm2. For the Cas9–RNA–DNA complex, the sizes of the ROIs for the REC, HNH and RuvC-PI domains that enclosed the whole region of each domain were about 13 × 10 nm2, 7 × 7 nm2 and 13 × 10 nm2, respectively. The 2D correlation coefficient was calculated frame by frame for each ROI. The 2D correlation coefficient is defined as,
in which
and , respectively. The
center positions of the HNH and REC domains were calculated, as
described below. First, the ROIs that enclosed the whole HNH and REC
domains were about 7 × 7 nm2, and then the center positions
of the ROIs were calculated by the X, Y, and Z data of the HS-AFM
images. The X and Y data correspond to the lateral coordinates, while
the Z data correspond to the height. After the conformational change in
the HNH domain, the identical ROIs were used for the calculation of the
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Data availability
References
