626 cryo transfer holder

Cryo-EM samples were prepared on continuous carbon coated C-flat holey carbon grids (Protochips). Grids were plasma cleaned for 10 s in air using a Solarus Plasma Cleaner (Gatan) operating at 10 Watts. Immediately following crosslinking, 4 μl of purified TFIID-IIA-SCP complex or TAF-less PIC was added to the plasma-cleaned grid and loaded into a Vitrobot (FEI). The sample was incubated on the grid for 10 minutes at 4 °C and 100% relative humidity to enhance its absorption onto the carbon substrate, then was blotted and immediately plunge-frozen in liquid ethane. Frozen grids were transferred to a 626 Cryo-Transfer Holder (Gatan) and loaded into a Titan electron microscope (FEI) operating at 300 keV. Images were recorded with a K2 direct electron detector (Gatan) operating in counting mode at a calibrated magnification of 37,879 (1.32 Å pixel⁻¹) and a defocus range of −2 μm to −4 μm, using the Leginon data collection software for semi-automated acquisition targeting. 20-frame exposures were taken at 0.5 s per frame (10 s total exposure time), using a dose rate of 8 e⁻ pixel⁻¹ s⁻¹ (4.6 e⁻ Å⁻² s⁻¹ or 2.3 e⁻ Å⁻² per frame), corresponding to a total dose of 46 e⁻ Å⁻² per micrograph.