Figure 3.

Modelling and other experimental evidence of ice nucleating protein (INP) interactions. (a) Tyrosine ladder interactions between INP monomers (InaZ) as predicted by FRODOCK using AlphaFold models, with tyrosine residues (in yellow) showing head-to-tail dimerisation. (b) Ice nucleation assay comparing INP preparations at 50 µg mL⁻¹ alone (grey dots) or combined with either 100 mM tannic acid (TA; blue dots) or 100 mM TA and BdAFP from cold-acclimated wildtype lysates (yellow dots). Significance (p < 0.01, one way ANOVA) compared to INPs alone is denoted by lowercase letters. Ice nucleation assays were repeated in triplicate with similar results. Note that the colligative depression of the TA solution was <0.2 °C. (c) An AlphaFold model of the P. syringae INP InaZ twisted beta-solenoid structure with the GPI-anchor and linker hidden, and the tyrosine ladder residues highlighted in yellow (top), shown together with a simplified illustration of the INP monomer with exposed tyrosine ladders represented by yellow bars (bottom). (d) InaZ with charged residues on the opposing side of the beta-solenoid to the tyrosine ladder, with negatively charged residues highlighted in red and positively charged residues in blue (top), shown together with a simplified illustration with charged residues represented by bars (bottom). (e) Schematic illustration of INP monomers (with the GPI anchor and cap sequence removed) organised by tyrosine ladder interactions, highlighted in yellow, to form a short INP filament. INP monomers would first partially dimerise with a single monomer forming tyrosine ladder interactions at each end of the twisted solenoid with two separate monomers on opposing sides. (f) Self-assembly of INP filaments would precede the assembly of parallel filaments into aggregate sheets, which could be stabilised through electrostatic interactions by the outward-facing positively and negatively charged residues (blue and red lines, respectively) found on opposing ends of the monomers on the side of the beta-solenoid opposite the tyrosine ladders (yellow lines). The schematic illustration shows 35 INP monomers (~250 nm² of surface area) arranged into a sheet that would form patches on the surface of P. syringae (top down view). It should be noted that, in theory, 34 INP monomers must interact to reach a critical embryonic ice nucleus mass needed to achieve high sub-zero ice nucleation at −2 °C (see Section 3 Discussion).