Fig. 2: The experimental system of U2AF2 (RRM1, 2) and a comparison of FRET efficiency histograms from seven different laboratories.
a, Schematic of the dynamics of U2AF2. The apo state (in gray, top) undergoes fast exchange between an ensemble of detached structures of which five representative structures are displayed. A slower exchange occurs between the dynamic detached ensemble and a compact conformation (PDB ID 2YHO) shown below. The holo state (in green, PDB ID 2YH1) bound to a U9 RNA ligand (in dark gray) assumes a well-defined, open conformation. Positions of cysteine mutations introduced for labeling (L187 in RRM1 and G326 in RRM2) are depicted as black spheres with the mean dye position determined by AV calculations indicated by red spheres. b,c, SmFRET efficiency histograms reported by the seven participating laboratories for apo (b) and holo (c) measurements of U2AF2. The top shows the individual FRET efficiency histograms and the bottom shows the average FRET efficiency histogram (solid line) with standard deviation (light area). d, SmFRET efficiency E histograms of U2AF2 in the apo state. The top shows a representative 1D FRET efficiency histogram with a Gaussian fit (laboratory 1). The middle shows the reported mean FRET efficiencies reported by seven laboratories. The mean value from all datasets is 0.739 ± 0.029, shown above with the corresponding standard deviation in gray. The bottom shows the extracted mean FRET values after reanalysis of the collected data. After reanalysis, the agreement improved to 0.742 ± 0.008. e, SmFRET efficiency histogram comparisons of U2AF2 in the holo state. 5 µM of U9 RNA was used to obtain the holo state. The top shows a representative 1D FRET efficiency histogram of laboratory 1 fitted to two Gaussian distributions to determine the FRET efficiencies of the different subpopulations, yielding mean FRET efficiencies of 0.44 for RNA-bound and 0.76 for the RNA-free conformation. The middle shows the mean FRET efficiencies reported by the seven laboratories. The mean values from all seven of the datasets were 0.45 ± 0.04 for the RNA-bound conformation (in green) and 0.78 ± 0.04 for the RNA-free conformation (in gray). The bottom shows the reanalysis of the holo measurements yielding values of 0.42 ± 0.02 and 0.77 ± 0.03 for RNA-bound and RNA-free fractions, respectively.
