Figure 2. USP30 employs a Cys-His-Ser catalytic triad.

a, Superposition of the USP30 diubiquitin complex (blue/orange) with the USP7~ubiquitin aldehyde complex (yellow/grey, PDB 1NBF⁴⁵). Close-up views focus on coordination of the distal ubiquitin C-terminus with hydrogen bonds as black dotted lines. Yellow arrows indicate lost hydrogen bonds in USP30 due to indicated amino acid changes. b, Fluorescence polarisation binding experiment of USP30^c13i to ubiquitin-KG-TAMRA, indicating weak binding. Experiments were performed in triplicate and errors represent standard deviation from the mean. c, Cartoon representation of the USP30^c13~Ub-PA complex and close-up view of the catalytic triad. d, Catalytic efficiencies (k_cat/K_M) for Ser477 mutant proteins derived from a ubiquitin-KG-TAMRA cleavage assay, see Supplementary Fig. 5e-f. Experiments were performed in triplicate and error bars indicate standard error. e, Lys6-diubiquitin cleavage assay for indicated USP30 mutants, performed in duplicate. Uncropped images for gels are shown in Supplementary Data Set 1.