Titan krios 300 kv microscope

Before vitrification, proteins were buffer exchanged to 25 mM HEPES (pH 7.5), 250 mM NaCl, 0.025% beta-octyl-glucoside, 1 mM DTT, and 1 mM Mg-ATP and concentrated to 1.2 (ATAD2 Walker B-H3/H4K5Q complex) or 1.8 mg/mL (ATAD2 Walker B mutant). Proteins were frozen on glow-discharged Quantifoil Cu R1.2/1.3 300 mesh grids with a Vitrobot IV (Thermo Fisher Scientific) set at 15 °C and 100% humidity and blotted with -10 or 0 blotting force with 3 s blotting time. For the ATAD2 Walker B -ATP dataset, 9187 movies were collected using a Titan Krios 300 kV microscope with a Gatan Bioquantum GIF/K3 direct detector varying the defocus from 0.8 to 2.2 µm with 0.5 µm step size at Harvard Center for Cryo-Electron Microscopy (HC2EM) (Table 1 and Supplementary fig. 2). For the ATAD2 Walker B-histone H3/H4-ATP dataset, 4,961 movies were collected using a Titan Krios 300 kV microscope with a Gatan Bioquantum GIF/K3 direct detector varying the defocus of 0.8 to 2.4 µm with 0.2 µm step at POSTECH IMP.

Purified MMTV STC sample (0.5 mg/ml) was applied to R1.2/1.3 gold UltrAufoil grids, Au 400 mesh (Quantifoil), and cryo-EM grids were prepared by freezing using a manual plunger at 4°C. The grids were clipped and subsequently stored in liquid nitrogen for future data acquisition. Data were collected at the Scripps Research Institute Cryo-EM facility, La Jolla using a FEI Titan Krios (300 kV) microscope equipped with a Gatan K2 summit direct detector. The complex was imaged at 22 500× magnification and the pixel size was 1.31 Å. Movies were collected in counting mode with an electron dose rate of 3.3 electron per pixel per second. The defocus range was −1.3 to −3.0 μm. A total of 1578 movies of 100 frames/movie were collected. The data collection parameters are presented in Supplementary Table S1.