F416 4k 4k cmos camera

Samples of 26S-bound Ubp6-UbVS were diluted to ~25 nM in 60 mM HEPES pH 7.6, 50 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 1 mM TCEP and either 1 mM ATP or 1 mM ATPgS (Sigma). A thin layer of carbon was applied to 400-mesh Cu-Rh maxtaform grids (Electron Microscopy Sciences) by chemical vapor deposition, and grids were subsequently exposed to a 95% Ar/5% O2 plasma for 20 seconds to glow-discharge/activate the carbon surface. Grids were pre-treated with 4 µl of 0.1% poly-L-lysine hydrobromide (Polysciences) to prevent preferred orientation of 26S particles on carbon. Poly-L-lysine solution was then wicked away, grids were washed with 4 µl of H₂O, and 4 µl of sample was applied. 252 and 357 images of negatively stained (2% uranyl formate) 26S-Ubp6-UbVS complexes in the presence of ATP or ATPgS, respectively, were collected at a nominal magnification of 52,000 × on an F416 CMOS 4K × 4K camera (TVIPS) with a pixel size of 2.05 Å/pixel at the sample level. Images were acquired on a Tecnai Spirit LaB₆ electron microscope operating at 120keV, with a random defocus range of −0.5 µm to −1.5 µm and an electron dose of 20e-/Ų. Data were acquired using the Leginon automated image acquisition software^{53 (link)}.