The effect of combined charged-based amino acid constraints and PSSM restrictions on deimmunization of human erythropoietin (PDB ID 1EER). Designs (n=1000) were generated with an mhc_epitope weight of 1.0 (red) and 0.0 (blue), and with an amino acid composition constraint weight of 0.5, after restricting designable residue identities to those with a base 2 log-odds score of 1, 2, or 3, or with no constraints (NC). The green dashed line indicates the wild-type value (relaxed, but with no design). The effect on six metrics is evaluated: (A) REF2015 score, (B) unweighted mhc_epitope score, (C) PackStat score, and the number of (D) buried unsatisfied hydrogen bonds, (E) positively charged residues, and (F) negatively charged residues. See also Figure S7 and Figure S8 for corresponding plots for other protein targets.