Extended Data Fig. 6: Trajectories, energy landscapes and inter-well transition rates for R-loop formation on the different T6 target sequences.
From: The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex
Shown are results measured on a single molecule with a, T6, b, T6-flipG, c, T6-flipT and d, T6-M17 target sequence. For each target are shown: a DNA untwisting trajectory in presence of Cascade (left, light colours represent raw data at 3947 fps, dark colours the data after 100-point adjacent averaging), the sequence of the particular target (with PAM bases in orange, the flipped base positions in purple and mismatches with the crRNA underlined in red), a histogram of the angular positions obtained from all trajectories of the given molecule (middle, top) as well as the corresponding free energy landscapes for R-loop formation (middle, bottom). In the latter, the apparent (black line), the deconvolved (line and scatter points) and the reconvolved (coloured line) free energy landscapes are shown. The apparent energy landscape in absence of Cascade (from which the deconvolution Kernel was obtained) is shown as a purple line. Inter-well transition rates from measured and simulated DNA untwisting trajectories are shown on the right. In the simulations, the R-loop length can be monitored directly from which correct transition rates of the simulations were calculated. Transition rates obtained by the three different ways show a good overall agreement. This validates the determination of realistic inter-well transition rate from experimental trajectories.
