Tecnai g spirit biotwin transmission electron microscope

Carbon-coated hexagonal mesh copper grids (Electron Microscopy Sciences) were negatively glow-discharged for 30–45 s at 15 mA. Purified recombinant hAire and mAire CARD variants were diluted in TNE buffer (100 mM Tris pH 8, 50 mM NaCl, 10 mM EDTA) to a final concentration of ~50–200 µg/ml and immediately applied to the glow-discharged grid (Electron Microscopy Sciences). After 30 s, sample was blotted away with Whatman paper and washed with water twice. The grid was then pre-stained with 0.75% uranyl formate (UF) for 5 s, blotted and stained with 0.75% UF for 30 s. Stained grids were blotted and aspirated dry. Grids were imaged using a FEI Tecnai G² Spirit BioTWIN transmission electron microscope (TEM). In order to obtain higher resolution images of refolded mAire CARD wild-type filaments, grids were imaged with a FEI Tecnai F20 TEM. Images collected on the F20 TEM were analyzed with the image-processing suite EMAN2^{67 (link)} and 2D-classification of the processed images was done with RELION 3.0^{68 (link)}.

Wild-type DREP2 CIDE and R36E mutant samples after affinity chromatography purification were diluted to 0.8 mg/mL to prepare the high concentration sample and 0.1 mg/mL to prepare the low concentration sample. DREP4 CIDE samples were also diluted to 1 mg/mL as the high concentration sample and 0.1 mg/mL as the low concentration sample. For negative staining, 10 μL of each protein sample was placed onto a glow discharged copper grid and stained with 1% uranyl formate at pH 4.5 for 30 s and air-dried. The grids were imaged using a Tecnai G² Spirit BioTWIN Transmission Electron Microscope (FEI Company, Hillsboro, OR, USA) and recorded with an AMT 2k CCD camera (Thermo Fisher Scientific, Waltham, MA, USA).