Superdex 200 pc 3.2 30

SAXS data on the SALM3 LRR-Ig and PTPσ complex was measured by the SEC-SAXS method at Diamond Lightsource on the B21 beamline. For SALM3 LRR-Ig and PTPσ complex preparation, SALM3 LRR-Ig and PTPσ were mixed in the molar ratio of 1:1.2 of SALM3 LRR-Ig (60 µM) and PTPσ (80 µM) in total volume of 50 µl, followed by incubation at 4 °C for 1 h. Sample compartment and exposure cell were cooled to 4 °C. Superdex 200 PC 3.2/30 (GE Healthcare) column was used with flow rate of 0.5 ml/min in 30 mM Tris HCl pH7.5, 150 mM NaCl. Data processing was performed using ScÅtter³⁸ and DATASW^{39 (link)}. Particle shapes at low resolution were reconstructed ab initio by DAMMIN^{40 (link)} in P1 with P(r) function generated from the data truncated to 0.25 Å⁻¹. A total of 10 independent reconstructions were performed, and the models were averaged with the program DAMAVER^{41 (link)}. Rigid body modeling was performed using CORAL^{42 (link)}, where available partial crystal structures were treated as rigid bodies while missing residues were modelled ab initio as a Cα-chain by the software. The final model was converted to a full all-atom model using FULCHER tool (Shkumatov A. et al., unpublished). The modeling was done with either P1 or P2 symmetry. Porod and dimensionless Kratky plots were calculated for estimation of D_max and the overall compactness of the models (Supplementary Fig. S3).

Five-hundred mL of conditioned medium (C.M.) of FLAG-tagged Wnt3a (FLAG-Wnt3a)-expressing L cells in serum-free condition were prepared. HEPES buffer, pH7.4 (final 10 mM); EDTA, pH8.0 (final 1 mM); and protease inhibitors (final 1 mM PMSF, 5 μg mL⁻¹ leupeptin, 10 μg mL⁻¹  peptstatinA, and 5 μg mL⁻¹ aprotinin) were added to the C.M. Debris was removed by centrifugation for 30 min at 14,000 × g, and the supernatant containing FLAG-Wnt3a was applied to an anti-FLAG M2 affinity gel (Sigma) column that had been equilibrated with 15 mM HEPES buffer, pH7.4-150 mM NaCl. The column was washed with 10 column bed volumes of the same buffer containing 0.1 mM n-Dodecyl-β-D-maltoside. The bound proteins were eluted with the same buffer containing 125 μg mL⁻¹ FLAG peptide (Sigma). The eluate was concentrated 10–30 times with a Microcon centrifuge filter unit YM-100 (Millipore). Further purification was performed by using Superdex200 (PC3.2/30) gel filtration chromatography in a Smart-system (GE Healthcare). The elution of protein was profiled at 280 nm, and the eluate was sub-fractionated into 40-μL fractions.