Figure 6.

Characterization of additional CB FRET sensors and FRET-restraint-based Markov-chain Monte Carlo (MCMC) sampling. (a) Average fluorescence lifetime observed for F1_DA, F28_DA, F73_DA, and F99_DA in the absence (solid bars) and presence (dotted bars) of GephFL. Data from three different batches of experiments are presented as mean values ± SD. (b) Distance distribution obtained from the two Gaussian distributed distances fit model of different FlAsH-labeled sensors in the absence (solid lines) and presence (dotted lines) of GephFL. In the case of F1_DA and F28_DA, the change in the low-FRET state upon interaction with GephFL is larger. (c) MCMC sampling of the fluorophore positions in CB based on the experimentally obtained FRET restraints (Table S15), connectivity restraints (Table S14), and the available structures for CB-SH3⁻ (PDB: 4mt7) and CB-SH3⁺ (PDB: 4mt6). The cyan density reflects the spatially accessible volume of CFP, whereas the green color shows the spatially accessible volume of FlAsH incorporated at residue 99. The conformational change in CB from the free to the bound state allows both fluorophores to sample a much larger volume. In the bound, open state the excluded volume due to gephyrin binding should be considered.