Cryo-EM sample preparation

For negative staining EM grids preparation, 5 μL of Pol III PTC complex sample were applied onto glow-discharged copper grids supported by a continuous thin layer of carbon film for 60 s before negatively stained by 2% (w/v) uranyl formate solution at room temperature. The grids were prepared in the Ar/O₂ mixture for 15 s using a Gatan 950 Solarus plasma cleaning system with a power of 15 W. The negatively stained grids were loaded onto a Thermo Fisher Scientific Talos L120C microscope equipped with a Ceta CCD camera and operating at 120 kV at a nominal magnification of 92,000x, corresponding to a pixel size of 1.58 Å on the specimen.

For cryo-EM grids preparation, 3 μL of the sample at a concentration of 0.7 mg/mL dialyzed PTC complexes were applied to freshly glow-discharged Quantifoil R 1.2/1.3 holey carbon grids. After incubation of 5 s at a temperature of 4 °C and a humidity of 100 %, the grids were blotted for 8.5 s in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H₂/O₂ mixture for 30 s using a Gatan 950 Solarus plasma cleaning system with a power of 5 W. The ø 55/20 mm blotting paper (TED PELLA) is used for plunge freezing.