Affinity-purification of native protein complexes from C. thermophilum

Ground frozen mycelium was thawed on ice in NB-Hepes buffer containing 0.1% (vol/vol) IGEPAL^® CA-630 or 1% (vol/vol) Triton^TM X-100, including SIGMAFAST complete protease inhibitor cocktail (Sigma Aldrich) at a ratio of approximately 1 ml buffer per gram of cells. The lysate was cleared (20.000 rpm at 4 °C for 30 min) and the Flag-TEV-ProteinA (FpA) tagged proteins were affinity-purified from the supernatant using 300 μl IgG-Sepharose suspension (IgG-Sepharose 6 Fast Flow; GE Healthcare). Upon washing, proteins were eluted by TEV protease in a 2.5 ml Mobicol column (MoBiTec) at 16 °C for two hours and subsequently incubated with 50 μl ANTI-Flag^® M2 affinity gel (Sigma Aldrich) at 4 °C for one hour. Bound proteins were eluted by use of 100 μg/ml Flag peptide (Sigma Aldrich) at 4 °C for 30–60 min. Eluates were separated by SDS-PAGE and analyzed via Colloidal Coomassie staining and mass spectrometry. For the detection of glycosylation of ctNsp1, an O-linked N-acetylglucosamine antibody (abcam cat. no. ab2739, 1:1000 in PBS + 0.05% Tween-20/5% milk) was applied. Sucrose gradient ultra-centrifugation was performed with the Flag eluates, which were loaded onto a continuous sucrose gradient (10–30% (w/vol) in NB-Hepes buffer), centrifuged at 26.000 rpm for 16 hours in a SW60 Ti swinging bucket rotor (Beckman Coulter) and fractionated.

To test different solvent compositions for ctNup53 and ctNup133, cryo-milled mycelium was dispensed on a 24-well format microtiter plate and subjected to a magnetic-bead based affinity capture procedure according to the protocol published by Hakhverdyan et al.³⁴. The formulations of extractants used for the experiment are presented in Supplementary Table 1. Mass spectrometric identification of proteins was carried out as described³⁴.