To select for sites to randomize in the CBX1 library to enhance its binding affinity toward H3K9me3, we used two different strategies. The first was to identify residues important for binding H3K9me3 compared with nonbinding peptides. Comparison of MIEC components of H3K9me3 binding to nonbinder components, along with avoiding choosing residues that were too conserved among chromodomains, we constructed a ranked list of candidate sites that showed the largest energetic difference between binders and nonbinders. The second strategy was essentially the same as the first strategy except that we selected sites generally important for CBX1 binding by comparing all CBX1 binder MIECs to a representative nonbinder (H3K27me3). Again, a conservation filter was applied to prevent selection of structurally important residues on the chromodomain (see Supplementary Methods and Materials for a more detailed description along with table S6).

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