Binding motifs, loop sequences and framework mutations of binders to ubiquitin-conjugating enzyme E2 R1 (CDC34). (A) Sequence motifs shared among binders of pre- and post-affinity maturation. There are two motifs for binders of both pre- and post-affinity maturation. Two sibling clones were isolated for motif 2 of post-maturation. The motif logo plots were generated by WebLogo [55]. In the logo plot, the following amino acid residues are in green: A, G, H, P, S, T. Amino acid residues of R, K, and N are in blue and the rest of the residues are in black; (B) Sequence alignment of eleven binders of post-maturation. Clone 2F12 (Red-squared) is the only sequence of the motif 2 and the rest of clones belong to motif 1. The FG loop of wild-type FN3 (WT-FN3) has eight residues, which was shortened to five residues in FN3 variants. Dashes are inserted to maximize the sequence alignment.