Fig. 1: Analysis of UFC1 T106 mutants.

a Super-positioning of UFC1 T106I (orange) onto WT UFC1 (PDB 2z6o) (blue) yields C-alpha atoms with an RMSD of 0.266 Å. Side chains of C116 and T/I106 are shown in stick representation. b A representative western blot showing the effect of UFC1 T106I, T106V, and T106A mutants on in vitro ufmylation (see Supplementary Fig. 1g for protein loading control). c The band corresponding to the ufmylated product (~40 kDa) was quantified for each mutant and normalized against the product formed in WT protein (data represented as mean ± SD, n = 5 independent experiments). Differences were significant for T106I, T106V (with p < 0.0001) and T106A (with p < 0.01, all calculated using two‐tailed unpaired Student’s t‐tests). d Western blot showing the effect of UFC1 T106I, T106V, T106A, and T106L mutants on ufmylation of substrate (RPL26) (see Supplementary Fig. 1h for protein loading control). Ufmylation reactions were performed in the presence of the 60S ribosomes and the above mutants. Data is representative of three independent experiments. e Superposition of the crystal structure of UFC1 T106A (red) with the WT protein (PDB 2z6o) (blue). The C-alpha of A106 is moved 1.8 Å, relative to the position of C-alpha of T106 in the WT protein. f The crystal structure of the T106A mutant shows that the side chain of E149 adopts two alternative conformations, one of which forms a hydrogen bond with the amide backbone of K108. Source data are provided as a Source Data file.