Small angle X-ray scattering

SAXS data were collected using the inline-SEC setup at the Australian Synchrotron SAXS/WAXS beamline. A co-flow setup was implemented to maximize signal-to-noise by allowing exposure of protein to high flux X-rays without direct contact with the capillary wall, while minimizing sample dilution^77,78. RIPK3:MLKL complex (unliganded or pre-incubated with Compound 10) was eluted from a Superdex 200 5/150 Increase column (Cytiva) in 200 mM NaCl, 20 mM HEPES pH 7.5, 5% glycerol at a flow rate of 0.2 mL/min, where the SEC eluate passes through a capillary in the path of the SAXS beam and scattering data were collected in 1 s exposures serially over the course of the chromatography run. Data were reduced, 2D radially integrated, and background scatter subtracted using ScatterBrain v2.82 (developed in-house by Stephen Mudie). The background scatter was calculated by averaging exposures from the SEC run prior to protein elution, and was subtracted from the scatter arising from the protein peak off the SEC column. Exposures from the apex of the protein elution peak were averaged and subjected to analyses using the ATSAS 3.0 package⁷⁹ including Guinier analyses in PRIMUS, P_r in GNOM, and comparisons with theoretical and experimental scatter using CRYSOL. Data collection and processing statistics are summarized in Supplementary Table ¹.