Quantitative evaluation of alternative threadings of TM4 and TM5 into the cryo-EM map. The eight most likely threadings of the TM4-TM5 hairpin, based on the analysis described in Fig. 4 A, are evaluated further. (A) The ensembles of models constructed for each threading are evaluated in terms of the evolutionary couplings between the TM4-TM5 hairpin and the outer helix of subunit c (Fig. 3 B). For clarity, a single, global score was calculated for each model from this evolutionary data (Materials and methods), and a histogram of that score across the ensemble is shown; by definition, the maximum value of this score (i.e., no distance violations) is 11. (B) Analogous analysis to A, instead based on Cα–Cα distances inferred from cross-linking experiments (Materials and methods and Table S2); by definition, the maximum value of this score is 19. (C) Analysis of the top three threadings according to A and B, in terms of the proximity between Glu58 in subunit c, Arg145 in TM4 of subunit a, and Gln201 in TM5 (see Results and discussion). The plots show the percentage of models in each ensemble for which the distance between these side chains is less than or equal to an increasing value between 0 and 20 Å.