Extended Data Fig. 7: Deconvolution of an apparent free energy landscape of R-loop formation exploiting the measured R-loop dynamics.
From: The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex
a, Apparent (black line) and deconvolved (red lines) free energy landscape of R-loop formation obtained for the T6-flipG target. Deconvolved energy landscapes were calculated for different entropy scaling factors b (see legend). The blue dashed line is a linear approximation of the apparent energy landscape between the local maximum at ~7 bp and the minimum at 26 bp. Increasing entropy scaling factors decrease the height of the free energy barriers between the local energy wells. To obtain a deconvolved free energy landscape that supports the measured dynamics of the R-loop formation as well, transition probability plots were used (see b). b, Transition maps revealing the dynamics of R-loop formation. Each transition map shows the probability distribution describing the length change of R-loops (vertical axis) of a given apparent length at time t (horizontal axis) after various time delays Δ\(t\) (2, 5, 10 and 25 ms). Shown are maps for the measurement in a (upper row) as well as for the Brownian dynamics simulations using the deconvolved energy landscapes obtained for different energy scaling values b and single base pair stepping rates kstep. While all b values well describe the intra-segment diffusion at 2 ms, significant differences are seen for the inter-segment dynamics at larger timescales, for which b=10 and kstep=7000 s−1 provide the parameter fit that best describes the measured data. c, Total residue (RMS) between simulated and measured transition plots (for all time differences) shown for different parameter sets of b and kstep. The RMS for a given parameter set was calculated from the sum of the RMS values at Δt = 2, 5, 10 and 25 ms). Coloured circles correspond to the 3 parameter sets highlighted/chosen in a and c. Across all targets we obtained a single base pair stepping rate of \({k}_{\mathrm{step}}=6000\pm 2000\,{s}^{-1}\) (s.d.).
