Figure 3. CT8 traps the TMD in a helical conformation with a defined orientation.

(A) Positions of single cysteine mutations (blue dashes) analyzed by BMH crosslinking. (B) BMH crosslinking reactions of 126-mers containing a single cysteine at the indicated positions. (C) Crosslinking intensities (NC × Sec61α) were quantified by phosphorimaging of the gels shown in (B) and plotted as a function of cysteine position. Inset, TMD α-helix model (red) highlighting positions with strongest crosslinks to Sec61α (yellow). (D) Overlay of BMH crosslinking profiles for the indicated constructs. Crosslinking intensities were normalized to an internal standard (Cys49 126-mer) included in each experiment. For raw phosphorimaging data, see Figure 3—figure supplement 1. (E) Cartoon depicting approximate TMD disposition (red) of 126-mer assembled in the presence and absence of CT8.

DOI: http://dx.doi.org/10.7554/eLife.01483.007

Figure 3—figure supplement 1. CT8 traps the TMD in a helical conformation with a defined orientation.

(A–D) BMH crosslinking profiles of RNCs of various lengths that carry single cysteines at the indicated positions of the TMD. The graphs on the right plot the intensity of the TMD/Sec61α crosslink (×Sec61α) against the position of the cysteine along the TMD. For part (D), the V41P position was not changed to cysteine. An internal standard construct (Cys49 126-mer) was included in each experiment (last pair of lanes in each gel series) to enable comparison of crosslinking efficiencies between different experiments. The values on the y-axes for plots in (A–D) are therefore comparable.

DOI: http://dx.doi.org/10.7554/eLife.01483.008