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. 2020 Mar 4;6(10):eaay7505. doi: 10.1126/sciadv.aay7505

Fig. 5. Distribution of mutagenic effects on the surface immunostaining and conformational stability of opsin and rhodopsin.

Fig. 5

Trends associated with the surface immunostaining of rhodopsin variants and their predicted energetic effects on cotranslational and posttranslational folding are shown. (A) Violin plots depict the statistical distribution of the effects of all possible missense mutations within TM2 (480 total, blue) and TM7 (460 total, red) on the energetics of rhodopsin folding in Rosetta energy units as calculated using the RosettaMembrane energy scoring function (25). The shape of each distribution was defined using a kernel smoothing function. Dashed lines within the violins reflect the median value, while dotted lines within the violins reflect the positions of the 25th and 75th percentiles. Positive ΔΔG values are indicative of a destabilization of the native structure. (B) Violin plots depict the statistical distribution of the effects of all possible missense mutations within TM2 (480 total, blue) and TM7 (460 total, red) on the translocon-mediated membrane integration of the corresponding transmembrane domains, which were calculated using the ΔG predictor (20). The shape of each distribution was defined using a kernel smoothing function. Dashed lines within the violins reflect the median value, while dotted lines within the violins reflect the positions of the 25th and 75th percentiles. Positive ΔG values indicate the translocon-mediated membrane integration of the helix is unfavorable. (C) Violin plots depict the statistical distribution of surface immunostaining values associated with missense mutations within TM2 (401 total, left) or TM7 (407 total, right) in the presence (dark blue, dark red) and absence (light blue, light red) of 5 μM 9-cis-retinal. Immunostaining intensity values were determined by deep mutational scanning and were normalized relative to the value for the WT protein. The shape of each distribution was defined using a kernel smoothing function. Dashed lines within the violins reflect the median value, while dotted lines within the violins reflect the positions of the 25th and 75th percentiles. Values reflect the averages from two biological replicates.