Fig. 4: Xtrapol8 enables successful extraction of the mEos4b red-off state.
The models in cyan and green are mEos4b in the red-on and red-off state, respectively, as downloaded from the PDB (PDB entry 6GP0 and 6GP1, with the only difference that features of green mEos4b were omitted) with the chromophore indicated in yellow (reference state, a, b) and magenta (triggered state, d–e). a traditional 2mFobs − DFcalc electron density after rigid body refinement of the red-on state model in the red-off data indicates the absence of signal for the red-off state. b q-weighted Fourier difference map (\({F}_{{{{{{\rm{obs}}}}}}}^{{{{{{\rm{red}}}}}}-{{{{{\rm{off}}}}}}}-{F}_{{{{{{\rm{obs}}}}}}}^{{{{{{\rm{red}}}}}}-{{{{{\rm{on}}}}}}}\)) superposed on the red-on model. c the difference-map method maximizes the information content of extrapolated difference maps and estimates the occupancy to be 0.325 (magenta dashed line). d–e Initial q-weighted 2mFextrapolated − DFcalc (d) and mFextrapolated − DFcalc (e) electron density maps calculated for an occupancy of 0.325 superposed on the red-off model. f The distance-analysis method, which approximates the correct occupancy by fitting a sigmoid to the evolution of interatomic distances as a function of all tested occupancies and then retrieves the occupancy value for which 99% of the plateau is reached, returns an occupancy estimate of 0.380.
