Fig. S2.

Conformation and conformational dynamics of RRM1,2-C187-C326. (A) Exemplary intensity recording of green and red photons after green and red excitation, respectively, over the course of a measurement. Right shows a zoom in of the measurement to display bursts of photons of individual molecules. (B) Exemplary biexponential fit to the donor lifetime histogram obtained from an MFD-PIE experiment of RRM1,2-C187-C326-Atto532/Alexa647. (C and D) For the following experiments, RRM1,2-C187-C326 was labeled with Atto532 and Atto647N. (C) FRET efficiency histogram of solution spFRET MFD experiments of RRM1,2 alone (gray; data from 31,659 molecules), in the presence of the strong Py-tract U9 (red; data from 38,481 molecules); and of the weak binding sequence U4A8U4 (blue; data from 18,057 molecules). (D) Two-dimensional histograms of spFRET efficiency versus donor lifetime from the same measurements as in C. Populations of static molecules are described by the polynomial static FRET line (Eq. S5; black line), whereas molecules undergoing conformational dynamics on the timescale of µs-ms deviate from this line (dynamic FRET curve in Eq. S7; red line). The fully open and closed conformations were determined from lifetime fits of the data and correspond to the intersections of the dynamic FRET curve with the static FRET line. (E) (Left) FRET efficiency histogram and (Right) 2D histogram of spFRET efficiency versus donor lifetime of RRM1,2-C187-C326-Atto532/Alexa647 in the absence (gray) and in the presence of A9 RNA (red) or A13ACAGG RNA (magenta). (F) (Left) FRET efficiency histogram and (Right) 2D histogram of spFRET efficiency versus donor lifetime of URRM1,2-C187-C326-Atto532/Alexa647/U2AF35(UHM) in the absence (gray) and in the presence of A9 RNA (red) or A13ACAGG RNA (magenta). (E and F) The static (black; Eq. S5) and dynamic (red; Eq. S7) FRET curves are shown.