Fig. 6: Structural characterization of U2AF2.
a, Structural flexibility of U2AF2 is given by translational (left) and rotational (right) movement of the two domains. Representative structures are taken from the ensemble determined using NMR and SAXS measurements29. b, Degeneracy of structural states in FRET measurements. The position of the two domains of U2AF2 is illustrated by the COM of the Cα atoms in RRM2 (residues 260–329, colored) with respect to RRM1 (residues 150–227, black) for the 200 structures of the conformational ensemble29. The COM of RRM2 is color-coded according to the FRET efficiency determined using AV3 calculations. c, A schematic of the kinetic model used for the global dynamic PDA of U2AF2 (Supplementary Note 17). d, Distance distributions obtained from a donor fluorescence decay analysis by a model-free MEM approach (Supplementary Note 15). The distance distribution from the NMR–SAXS ensemble29 (light blue) was used as the prior distribution. The expected interdye distances for the compact apo and open holo states are shown as red and blue dashed lines (PDB 2YH0 and 2YH1). Shaded areas indicate the distance broadening due to the flexible dye linkers of 6 Å. The distribution in the donor–acceptor distance RDA for different dye pairs is shown. e, Filtered-FCS reveals conformational dynamics in the U2AF2 apo ensemble on two timescales, tR,1 = 9 ± 3 and tR,2 = 300 ± 90 µs, average and standard deviation (n = 3, results from laboratory 1 are shown). The two species were defined at the lower and upper edge of the FRET efficiency histogram shown in Fig. 2b, top panel (see Methods and Supplementary Note 16 for details). The species autocorrelation functions (SACFs) and one of the two species cross-correlation functions (SCCFs) are shown. The weighted residuals are shown above. f, The PDA analysis was conducted globally over both apo (top) and holo (bottom) measurements using time windows of 0.5, 1.0, 1.5 and 2.0 ms (the 1.0 ms time window histograms are shown). A relaxation time of roughly 10 ms for the dynamics between the detached ensemble and compact apo state with a small amplitude was determined (orange curve) (Supplementary Fig. 16 and Supplementary Note 17).
