Fig. S2.

(A) An example of a low-force (15 pN) unfolding trajectory. (B) Histogram of the folded state calculated form the trajectory in A. (C) Free energy profile $F\left(x\right)=-k_{B}T\log P\left(x\right)$. If $x_L$ is the distance to the transition state, then the transition state should be located at $\approx 2.34$ nm. The states corresponding to 2.34 nm are visited multiple times before the single unfolding event but are always followed by the visit to the folded state. Therefore, $P_{fold}$ calculated from the ensemble of trajectories starting at $x_L$ is near 1. From the histogram in B, we find that $x_L$ is visited 231 times before unfolding, which yields $P_{fold}\ge 0.996$.