Characterizing the Cy mutation. A) negGFP native gel assay of ANKS3-SAM, ANKS6-SAM wt and ANKS6-SAM R823W. A titration series with a constant amount of ANKS3-SAM and increasing amount of ANKS6-SAM R823W is unable to restore the ANKS3-SAM/ANKS6-SAM interaction. B) The Cy mutation (R54) according to our numbering is highlighted on the ANKS6-SAM crystal structure in orange. R54 forms salt bridges and hydrogen bonds with nearby atoms to stabilize the fold in this part of the domain. C) CD spectra of ANKS6-SAM wt and ANKS6-SAM R823W. The reduced CD signal at 222 nm and the shifted minima around 208 nm correlate with a loss of alpha-helical character and gain of random coil. D) Thermal denaturation curves of ANKS6-SAM wt and ANKS6-SAM R823W monitored by CD signal at 222 nm. ANKS6-SAM wt exhibits a broad, weakly cooperative unfolding curve with Tm approx. 48°C, while ANKS6-SAM R823W unfolding is completely uncooperative.