Extended Data Fig. 6: Functional studies of Sec18.
a, Schematic of purification of Sec18. b, SDS-PAGE gel of final SEC fractions and the fractions that were pooled for Cryo-EM studies of the Sec18 complex and the vacuolar fusion assay. c, Corresponding SEC trace showing the Sec18 oligomer eluting at expected volume. d, Native gel of final sample showing two oligomeric species. e, Representative slices of Sec18 complex under the hydrolyzing condition, subjected to 3DVA revealing split hexamer class. f, Alignment of all seven protomers, where each protomer is colored uniquely. g, Cartoon depicting the large pore in the heptameric state of Sec18 complex under the hydrolyzing condition. h, Cartoon of the vacuolar fusion assay (Methods) used to determine the function of the same Sec18 sample used for Cryo-EM studies of the y20S complex. Fusion only occurs when Sec18 prepares SNAREs for fusion, leading to FRET between the entrapped proteins from each fusion partner. i, Representative traces of fusion activity. j, Dot-plots of all activity assays, error bars denote SEM and line denotes mean (n = 3). A one-way ANOVA was performed, comparing all groups to the no Sec18 condition. ** represents a p-value less than 0.01.
