Computational design of a versatile, zero-radius proximity labeling enzyme

• Supplementary Data 2. 2D Flow cytometry data for all LplA variants tested. Data are shown for (a) LplA orthologs, (b) ESM point mutants, (c) ProteinMPNN point mutants, (d) ESM combined mutants (E3-21), Directed evolution variant (D2), MPNN active- site redesigns (M1-24) and final combined engineering variants (F1-30). The black line in each plot shows the shape of the template (W37V LplA). • Supplementary Data 3. Confocal microscopy of all ESM1b/1v predicted point mutations. mCherry-tagged LplA variants were imaged live in HEK 293T cells. All mutations were tested on the W37V-background as denoted by the superscript “V”. • Supplementary Data 4. SEC-SAXS Elution profiles and Oligomer fits for LplA variants. Each document shows elution profile and associated SAXS scattering curves with Oligomer- predicted model fits. Data are plotted in both Log(I) vs q and Kratky plot format to better emphasize relevant differences, as well as the residual (data – model) for each fit. • Supplementary Data 5. LplA structures used for SEC-SAXS Oligomer analysis. PDB files for all LplA structural models used for Oligomer analysis of the SAXS data. • Supplementary Data 6. Code used to analyze SEC-SAXS data.