Extended Data Figure 10. Validation of the PlxnD1 mutant.

(a) Negative stain 2D class averages of PlxnD1 WT were obtained by classifying 1305 particles into 10 classes. Scale bar represents 10 nm. (b) Negative stain 2D class averages of PlxnD1 mutant were obtained by classifying 1357 particles into 10 classes. (c) The double mutant of PlxnD1 was labelled with a thiol-reactive fluorescent dye, Alexa Fluor 488 C5 maleimide. The degree of labelling shows that the vast majority of PlxnD1 mutant molecules form the disulphide linked bond and thus the ring of the majority of PlxnD1 mutant molecules appears to be locked by the covalent bond. The degree of labelling for the hen egg ovalbumin, which we used as a positive control, is close to the number of free cysteines in ovalbumin; n=3. The data represent mean±SEM. P values were calculated by two-tailed t-test in Graphpad Prism, ***p<0.001