Extended Data Fig. 7: Mutational analysis of the SfSTING cross-filament contacts.
From: Cryo-EM structure of an active bacterial TIR–STING filament complex
a, Raw plate reader NADase assay measuring fluorescence increase over time as a proxy for NAD+ degradation. At 1 μM enzyme, WT* and E95Q* mutant SfSTING consume the ε-NAD substrate within the deadtime of the experiment (flat maximal signal). Error bars indicate standard deviation for average of three technical replicates. ΔA36–K41, Q279E, V280D, E290K, R307E, and D110A* (grey) show no activity at this enzyme concentration, and lines are obscured by other inactive construct curves. D110A* indicates that this is not a filament contact mutant but an intradimeric contact mutant. b, Bar-graph representation of NADase activity for mutant D110A similar to presentation in Fig. 4a. NADase activity is measured as fluorescence intensity at 5 min. Each bar within a set corresponds to 0.1, 1, and 10 μM enzyme. Baseline threshold indicates background fluorescent signal. Error bars indicate standard deviation for average of three technical replicates. Data representative of three independent biological replicates. One-way ANOVA comparison of mutant to WT at the same protein concentration with statistically significant mean values (P < 0.0001) marked by * or otherwise are greater than P = 0.05 and considered not significantly different. c, EMSA showing radiolabelled c-di-GMP binding to WT, D110A, and V280D mutant SfSTING. Note: lack of probable filament band for the V280D mutant. Image representative of n = 2 experiments. d, EMSA showing radiolabelled c-di-GMP binding to all mutants. Note: lack of probable filament band for majority of mutants, consistent with lack of observed filaments by electron microscopy. e, Extended negative-stain EM micrographs for all mutants in the presence of c-di-GMP. Scale bar represents 100 nm. Images representative of n ≥ 3 experiments. f, Extended plate reader growth curve assay data for most tested mutants. WT* and GFP* curves are identical to Fig. 4c. All curves represent the mean of 3 technical replicates. Data representative of >2 independent biological replicates. g, Plate reader growth curve assay for additional mutants V280D and D110A. All curves represent the mean of 3 technical replicates. Data representative of >2 independent biological replicates.
