Figure 2: EPR spectroscopy.

(a) The first derivative absorption cw-EPR spectra of PROXYL spin-labelled detergent-solubilized NavMs constructs are depicted in coloured lines, with A221C and A223C spin-labelled mutants in a liposome environment overlaid (black lines) in the top two panels. Additional shoulders present in the low-field region for A223C and I241C are indicated by ‘*’. In the bottom panel, the spectrum of the A221C/D250C double mutant (red line) is compared to the 1:1 summation (black line) of the spectra of the A221C and D250C single mutants. Spectra were normalized to reflect approximately equal numbers of spins. (b) Background-corrected dipolar evolution data (black lines) and the fits to the DEER data obtained by Tikhonov regularization (coloured lines). (c) Distance distributions obtained by: Tikhonov regularization (different coloured lines for each mutant spectrum), except for the A221C–D250C double mutant (bottom panel) where two Rice distributions were used; MMM predictions based on the first static model structure are represented as orange lines and those based on the first dynamic model are thick grey lines; MMM predictions based on the final dynamic model are in black dotted lines. All plots are normalized by amplitude. (d) Three-dimensional structure of the NavMs transmembrane pore crystal structure with the CTD as determined by the DEER spectroscopy/molecular dynamics approach.