Fig. 3. Distance measurements on HiSiaP.

a Difference distance map of HiSiaP in the open (PDB-ID: 2CEY²⁹) and closed (PDB-ID: 3B50¹³³) conformation¹²⁵. The dark spots mark protein regions that undergo large conformational changes relative to each other. The double variants for distance measurements are highlighted with circles. b Surface presentation of HiSiaP (grey) in the open (left) and closed (right) conformation. The accessible volumes of the spin label at six different labelling positions were calculated with mtsslWizard and are represented by magenta meshes. Accessible volumes of FRET label maleimide-Alexa Fluor 647 were calculated with FPS⁶⁷, and are shown as blue meshes. The double variants that were used for experiments are identified with coloured lines, corresponding to the circles in (a). c Distance measurements with four different double variants of HiSiaP without (−) and with (+) substrate. The PELDOR/DEER results are shown above (grey curves for simulation, black curves for experiment) and the FRET distances below the x-axis (grey bars for simulation, black bars are the mean of n = 3 independent experiments). Raw data for all experiments are provided in the Supplementary Information. The red shade around the PELDOR/DEER data is the error margin calculated using the validation tool of DeerAnalysis⁵². The underlying principle of the validation tool is explained in the discussion section below. The red shades around the experimental smFRET distances are error bars based on the standard deviation of n = 3 independent experiments. Source data are provided as a Source Data file.