Structural Analysis of KAP1 Protein

KAP1 Fl was diluted to 0.05 mg/ml in 20 mM Hepes, pH 7.5, 300 mM NaCl, and 2 mM TCEP and cross-linked with 0.1% glutaraldehyde for 2 h at 23°C. The reaction was stopped by addition of 100 mM Tris–HCl, pH 7.5. The sample was diluted 20 times, and adsorbed to a glow-discharged carbon-coated copper grid (EMS, Hatfield) washed with deionized water and stained with a solution of 2% uranyl acetate. The grids were observed using an F20 electron microscope (Thermo Fisher Scientific) operated at 200 kV. Digital images were collected using a direct detector camera Falcon III (Thermo Fisher Scientific) with 4,098 × 4,098 pixels. The magnification of work was 29,000× (px = 0.35 nm), using a defocus range of −1.5 to −2.5 μm. After manual picking of 400 particles, Relion (105 (link)) was used to sort them into 2D class averages.