Prp2 mediated spliceosome remodeling creates a binding site for Cwc25 near the BP. (a) Schematic of our SiMPull experiment to test for binding of Cy5-tagged Cwc25 near the BP of spliceosome-associated pre-mRNA containing a single active Cy3 fluorophore. (b) FRET probability distribution of the raw single molecule FRET trajectories under B* conditions (in the presence of Prp2-mediated remodeling). (c) FRET probability distribution of the raw single molecule FRET trajectories under Bact conditions (in the absence of Prp2-mediated remodeling). (d) Representative single molecule FRET trajectories showing the binding and associated FRET between the Cy5 on Cwc25 and the Cy3 near the pre-mRNA BP under B* conditions with the raw donor (Cy3, green), acceptor (Cy5, red), and FRET (black) trajectories. Arrows indicate binding events with close proximity to the BP. (e) Representative single molecule FRET trajectory showing the absence of FRET between the Cy5 on Cwc25 and the Cy3 near the pre-mRNA BP under Bact conditions with the raw donor (Cy3, green), acceptor (Cy5, red), and FRET (black) trajectories.