Extended Data Fig. 6: Structure of CCP5 with a βII peptide analog with a glutamate branch at E435 shows no density for the peptide mainchain.

a. Structure of the Glu-P-peptide 2 transition state analog. b. CCP5 active site bound to the Glu-P-peptide 2 analog shows density only for the branch and branch-point glutamates. Scissile branch glutamate, pink, branch-point glutamate, cyan. |F_o| – |F_c| density before the addition of the analog shown as black mesh and contoured at 3.0σ. c. CCP5 active site bound to the Glu-P-peptide 2 analog colored as in (b). |F_o | – |F_c| density before the addition of the analog shown as black mesh and contoured at 2.0σ. No continuous density can be seen for the peptide mainchain. For reference, the position of the Glu-P-peptide 1 analog is shown in the active site in grey ball-and-stick representation. Data for three Glu-P-peptide 2 complex crystals were collected and none showed density for the peptide mainchain. d. Superposition of CCP5 in the CCP5:Glu-P-peptide 1 crystal structure (transition state complex; light grey) with class #1 CCP5:microtubule structure (substrate binding complex; color-coded as in Fig. 2) in which we modeled the βII-tubulin Phe436 at position +1. In the substrate bound structure R303 (green) is displaced relative to the position it occupies in the transition state complex structure (light grey). In the latter, the R303 side chain coordinates the phosphonate. In that conformation it would clash severely with the bulky Phe sidechain at the +1 pos. of the substrate. Cryo-EM map for the β-tubulin tail and CCP5 R303 sidechain contoured at 4.3σ.