Supplementary Figure 6: His-Lys6-Ub3 preparation.
From: Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30
(a) Schematic drawings of His-Lys6-Ub3 preparation. His-Lys6-Ub3 and His-Lys6-Ub3 (R74Adist) were enzymatically synthesized by E1, UbcH7 and NleL. Lys48 of all the Ub molecules was mutated to Arg to prevent the elongation of Lys48-linked chains. Lys6 of His-Ub and His-Ub (R74A) was mutated to Arg to prevent the elongation of Lys6-linked chains on the distal end of His-Ub. See also online methods. (b) SDS-PAGE of individual reaction steps in His-Lys6-Ub3 preparation. The lanes (i)–(iv) correspond to the steps (i)–(iv) shown in (a).
