Figure 4: MultiSTEP enables measurement of variant effects on FIX post-translational modification.

a. Factor IX domain and chain architecture. Signal: Signal peptide. Pro: Propeptide. Gla: Gla domain. EGF1: Epidermal growth-like factor 1 domain. EGF2: Epidermal growth-like factor 2 domain. Activation: Activation peptide. Protease: Serine protease domain. b-c. Heatmaps showing carboxylation-sensitive FIX-specific carboxylation scores (b) or carboxylation-sensitive Gla-motif carboxylation scores (c) for nearly all missense FIX variants (n = 2 replicates). Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased antibody scores). Black dots indicate the WT amino acid. Missing data scores are colored gray. Furin cleavage site (F), ω-loop (ω), ExxxExC motif (E), and aromatic stack (AS) are annotated above (b) and (c). For higher resolution heatmaps on the propeptide and Gla domains of FIX, please refer to Extended Data Fig. 7d–i. d-e. Density distributions of carboxylation-sensitive FIX-specific (d) or carboxylation-sensitive Gla-motif (e) carboxylation scores for FIX missense variants (orange) and synonymous variants (blue). Dashed line denotes the 5th percentile of the synonymous variant distribution. f. Scatter plot of median MultiSTEP-derived carboxylation-sensitive FIX-specific carboxylation scores and light chain secretion scores at each position in FIX. Points are colored by domain architecture, using the same color scheme as a. Black dashed line indicates >0.2 point deviation threshold from perfect correlation between carboxylation and secretion scores. Points with deviation greater than this threshold are labeled with their corresponding FIX position. Pearson’s correlation coefficient is shown. g. Crystal structure of FIX Gla domain (positions 47–92)⁹. Disulfide bridges (yellow) and γ-carboxylated glutamates are shown as sticks. Calcium ions are shown as teal spheres. Residues are colored as the ratio of the median carboxylation-sensitive FIX-specific carboxylation score to median FIX light chain secretion score. Missing positions are colored gray.