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. Author manuscript; available in PMC: 2025 Dec 13.
Published in final edited form as: Nat Struct Mol Biol. 2025 Jun 13;32(10):2099–2111. doi: 10.1038/s41594-025-01582-w

Figure 2: 17,927 MultiSTEP-derived secretion scores for 8,964 factor IX variants.

Figure 2:

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 FIX heavy chain secretion scores (n = 3 replicates) (b) or FIX light chain secretion scores (n = 2 replicates) (c) for missense FIX variants. Heatmap color indicates secretion score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased scores). Black dots indicate the WT amino acid. Missing data are colored gray. d-e. Density distributions of heavy chain (d) or light chain (e) secretion scores for FIX missense (orange) and synonymous (blue) variants. Dashed line denotes the 5th percentile of the synonymous variant distribution. f-g. Scatter plots comparing MultiSTEP-derived heavy chain (f) or light chain (g) secretion scores for seven different FIX variants (p.C28Y, p.A37T, p.G58E, p.E67K, p.C134R, p.S220T, and p.H267L), WT, and an unrecombined negative control to the geometric mean of Alexa Fluor-647 fluorescence measured using flow cytometry individually (n = 3 replicates). Error bars show standard error of the mean. The p.E67K missense variant is not present in (g). h. Scatter plot of median MultiSTEP-derived heavy chain (n = 3 replicates) and light chain (n = 2 replicates) secretion scores at each position in FIX. Points are colored by chain architecture, using the same color scheme as (a). Black dashed line indicates perfect correlation between secretion scores. Pearson’s correlation coefficient is shown. Gray background indicates <0.3 point deviation from perfect correlation. Points with median positional scores outside gray background are labeled with their corresponding FIX position. i. AlphaFold2 model of mature, two-chain FIX (positions 47–191 and 227–461). Putative FIX heavy chain (purple) or light chain (green) epitope positions shown as colored surfaces. j. Magnified view of the putative light chain epitope within the EGF1 domain (orange). Points colored in concordance with (h). k. Magnified view of the putative heavy chain epitope within the FIX serine protease domain (yellow). Points colored in concordance with (h).