Poly l lysine hydrobromide

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)}.

For grid preparation and data acquisition, 3.5 μL of 0.2 mg/mL purified Pol II(G) complex was applied to the glow-discharged C-flat R2/2 grid coated with a home-made continuous thin layer of carbon. In order to overcome an orientation bias, the grid was treated with 5 μl of 0.1% (w/v) poly-L-lysine hydrobromide (Polysciences) prior applying the sample. Excess sample was manually blotted and vitrified in liquid ethane. The entire procedure was carried out at 4 °C and 98% relative humidity. Cryo grids were loaded into a Titan Krios transmission electron microscope operating at 300 keV. Images were automatically acquired with Leginon³⁷ at a nominal magnification of 22,500X (1.31 Å/pixel at the specimen level) using a total dose of ~40 electrons per Ų and a nominal defocus range of 1.5 – 2.8 μm. A total of 2,710 images were acquired using a Gatan K2 Summit direct electron detector, operated in electron counting mode. Each image was acquired as a 35 frame dose fractionated movie over a 7 s exposure time.