Effect of phosphomimetic mutations on assembly properties of Shs1-containing complexes. Purified septin complexes containing four different Shs1 mutants were diluted into low salt and examined in the EM. (A–D) Mutant P1, Shs1(T6D; A); mutant P2, Shs1(T6D, T386D, S416D, S441D, and S447D; B); mutant P3, Shs1(T6D, T386D, S416D, S441D, S447D, S460D, T462D, S519D, S520D, S521D, S522D, S525D, and S545D; C); and mutant P4, Shs1(S259D; D). Micrographs depict representative images. Schematic diagrams on the left indicate the order of septin subunits (Shs1, magenta; Cdc12, green; Cdc3, black; Cdc10, blue), and the positions of each phosphomimetic mutation are shown as yellow stars.