Extended Data Fig. 7: Molecular dynamics simulations indicate the movements of the side chains of Ub^R42 and Ub^R72.

a–d, Wild-type ∆NC SdeA and indicated mutants were incubated with Ub, RAB33B and NAD⁺ at 37 °C for the indicated amounts of time. The samples were analysed using Coomassie staining and Pro-Q diamond phosphoprotein staining. Similar results were obtained in three independent experiments. Uncropped blots and gel images are shown in Supplementary Fig. 1. e, The structure of the SdeA mART–Ub–NADH complex. SdeA mART is shown as an electrostatic surface potential model. White, blue and red indicate neutral, positive and negative surfaces, respectively. Shown in green mesh is the 2F_o−F_c electron density map contoured at 1σ around the NADH molecule. f, Galactose-inducible pYES2 plasmids containing wild-type ∆NC SdeA or the mutants were transformed into yeast W303 strain. Five microlitres of cells in three tenfold serial dilutions were spotted on both glucose- and galactose-containing plates lacking uracil for two days before image acquisition. g, Purified ADPR-Ub proteins were treated with or without wild-type ∆NC SdeA. The samples were analysed using Coomassie staining and Pro-Q diamond phosphoprotein staining. h, Purified ADPR-Ub protein was subjected to top-down LC–MS analysis. The results indicated 100% ADPR-Ub. i, Wild-type ∆NC SdeA or other mutants were incubated with RAB33B and the prepared ADPR-Ub verified in g and h. The samples were analysed using SDS–PAGE, with Coomassie staining and Pro-Q diamond phosphoprotein staining. f–i, Similar results were obtained in three independent experiments. g, i, Uncropped blots and gel images are shown in Supplementary Fig. 1. j, k, The time series for the r.m.s.d. of the non-hydrogen atoms of the protein–ligand complex (j) and the ligand (k) in the SdeA mART–Ub–intermediate and SdeA mART–Ub–NAD⁺ systems during molecular dynamics simulations. These two plots indicate that both systems have reached equilibrium during the 200-ns simulations. l, m, The time series for the shortest distance between the NH1/2 atom of Ub^R72 and C1D of the ligand (l) and the distance between the NH1/2 atom of Ub^R42 and C1D of the ligand (m) in the two systems during molecular dynamics simulations.