Cryo-EM sample preparation of HAdV-F41

Purified HAdV-F41 was used at 4.2 mg/ml (pH 7.4) and 1.6 mg/ml (pH 4.0). The recombinant HAdV-F41 PB (fPB) was purified as described before (29 (link)) and used at 1 mg/ml in PBS buffer, supplemented with 5% glycerol. A HAdV-F41 sample at pH 4.0 was prepared by adding 2 μl of a 0.5 M citric acid/1 M Na₂HPO₄ (pH 3.4) solution to 25 μl of HAdV-F41 (pH 7.4), followed by incubating on ice for 15 min. Samples were vitrified on QUANTIFOIL Cu R200 2/2 (Electron Microscopy Sciences, catalog no. Q2100CR2) and QUANTIFOIL Cu R200 1.2/1.3 (Electron Microscopy Sciences, catalog no. Q3100CR1.3) grids for the virus particles and the recombinant protein, respectively. Before sample application, the grids were glow discharged using a PELCO easiGlow device (Ted Pella Inc.) at 15 mA for 30 s. Sample was applied by transferring 3 μl of sample onto the glow-discharged side of the grid, blotted, and plunge-frozen in liquid ethane, using a Vitrobot plunge freezer (Thermo Fisher Scientific), with the following settings: 22°C, 80% humidity, a blot force of −20, and a blotting time of 3 s. For HAdV-F41 at pH 7.4, sample was applied twice with a blotting step, using the same settings as above, between applications (51 (link)).