Minimal length requirement for the linker between the E1 docking motif and the E2 core domain in Ubc12. a, Schematic diagram of insertion and deletion mutants used for these experiments. b, Sequences of Ubc12 linkers (residues 14–26 in wild-type Ubc12) for insertion and deletion mutants used for these experiments. Insertions and amino acid changes are highlighted in red, and deletions are denoted by dashes. c, kcat Ubc12 variant/kcat wild-type Ubc12 for the 1, 4, and 7 residue insertion mutants (In1, In4, In7), the 1, 4, 5, 6 and 7-residue deletion mutants (Δ1, Δ4, Δ5–1, Δ5–2, Δ6–1, Δ6–2, Δ7) and an additional alanine-containing mutant to control for sequence requirements (Δ4A3), as indicated. The inset shows Ubc12~NEDD8 thioester formation for wild-type Ubc12 and the 7-residue deletion mutant, Δ7, at concentrations of 0.05, 0.1, 0.2, 0.5, 1 and 5 μM.