Sulfo lc sda sulfo nhs lc diazirine

For cryo-EM, phosphorylated NTSR1 was mixed in an equimolar ratio of βarr1^ΔCT at a concentration of ~5 μM, supplemented with NTS_8–13 to 10 μM final and incubated at 25 °C for 30 minutes before being concentrated with a 50 kDa MWCO concentrator (Amicon or Vivaspin) to ~350 μL and purified by size-exclusion chromatography using two tandem Superdex 200 increase 10/300 GL columns (GE healthcare). The mobile phase used was 20 mM HEPES pH 7.4, 100 mM NaCl, 0.00075% LMNG, 0.00025% GDN 0.0001% CHS and 0.2 μM NTS_8–13. Fractions containing complex were combined and diluted to 1 μM final and sulfo-LC-SDA (sulfo-NHS-LC-Diazirine) (sulfosuccinimidyl 6-(4,4’-azipentanamido)hexanoate) (Thermo Fisher), as a solution in DMSO, was added to 250 μM final and such that the final DMSO concentration was below 4%. The reaction was allowed to proceed for 45 minutes at 25°C in the dark, before hydroxylamine was added to a final concentration of 3 mM and incubated for an additional 15 minutes. The sample was distributed into a clear 96-well plate (90 μL/well), put on ice and irradiated for 45 minutes using a UVL-56 lamp. The sample was then pooled and concentrated to ~500 μL then re-run by SEC, again using two tandem Superdex 200 increase 10/300 GL columns (GE healthcare). Peak fractions were combined and concentrated with a 100 kDa MWCO concentrator (Amicon) to a final concentration of 4.5 mg/mL.