All proteins except SNX5 PX domain mutants were expressed in Escherichia coli Rosetta cells, whereas mutant constructs were expressed in BL21 Codon Plus supplemented with appropriate antibiotics. Single colonies from cultures grown on LB agar plates were inoculated into 50 mL LB2+ with ampicillin (0.1 mg/mL) and chloramphenicol (0.1 mg/mL), and grown at 37°C with shaking overnight. The following day, 30 mL from the overnight culture was used to inoculate 1 L LB media containing ampicillin (0.1 mg/mL) and chloramphenicol (0.1 mg/mL) and incubated at 37°C. Cells were grown to an optical density (OD) of 0.5–0.6 at 600 nm and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (except for the SNX5-IncE fusion, where expression was induced at OD600 of 0.8 with 1 mM IPTG). Cultures were incubated with shaking overnight at 18°C until the cells reach an O.D of 3.0 (~24 hr). Cells were harvested using a Beckman rotor JLA 8.1000 at 4000 RPM for 30 min at 4°C. Pellets were resuspended in 10 mL lysis buffer (50 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/ml benzamidine, 0.1 mg/ml DNase) per litre of culture. The cells were subjected to cell disruption and centrifugation at 18,000 RPM for 30 min at 4°C. The soluble fractions were first purified using affinity chromatography with glutathione-sepharose, and when required the GST tags were cleaved by thrombin while still bound to the column. The proteins were eluted in 50 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, and 1 mM DTT, and then further polished using gel filtration chromatography (Superdex 200, GE healthcare) in a buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl. The fractions corresponding to the respective proteins were then pooled and used directly for ITC or were further concentrated for crystallization.