Extended Data Fig. 3: Generation and functional validation of a H2-D^b yeast peptide–MHC library.

a, Schematic of the mouse class I MHC H2-D^b displayed on yeast as β2m, α1, α2 and α3 with peptide covalently linked to the MHC N terminus. b, Design of the peptide library displayed by H2-D^b. Design is based on the structure of the 6218 TCR bound to H2-D^b-restricted acid polymerase peptide 224–233 (SSLENFRAYV, D^bPA₂₂₄) (RCSB Protein Data Bank accession 3PQY)¹⁹. c, Mutation required for proper folding of the H2-D^b displayed on yeast (α2-W131 to α2-G131). Mutations were derived from error-prone mutagenesis. d, Design for two different lengths of H2-D^b libraries. For the nine-amino-acid (9 MER) library, residues from P1 to P9 were randomized, with limited diversity at MHC anchor positions P5 (Asn, N) and P9 (Met, Ile and Leu, M/I/L). For the ten-amino-acid (10 MER) library, residues from P1 to P10 were randomized, with limited diversity at MHC anchor positions P5 (Asn, N) and P10 (Met, Ile and Leu, M/I/L). TCR contact residues are coloured pink and MHC anchor residues are coloured red or blue. e–g, Selection of PA₂₂₄-H2-D^b error-prone library with 6218 soluble TCR. Increased MYC expression among induced yeast peptide-H2-D^b error-prone library at different rounds (RD1–RD4) of selection (e, g) and 6218 soluble TCR tetramer staining on the post-RD4 error-prone H2-D^b library (g). 6218 TCR was screened on the error-prone yeast library once.