Traces with potential dynamic behavior, identified by having distinct anticorrelated signals, were marked and analyzed individually. Step events were classified by having large anticorrelated changes in the donor and acceptor signal within the period of one to three frames (0.2 to 0.6 s) and were manually annotated. Force traces were smoothed using a median filter over five frames. Ramp events were classified by having more gradual anticorrelated changes and were manually marked. Ramp traces were then converted to the force domain, and only changes in load between 2 and 7 pN were fit. Dynamic events were only accepted if the acceptor was confirmed to be active and could not be accounted for by either donor or acceptor photobleaching. Very few dynamic-like sensors were observed under no-load conditions (table S5).

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