Cryo-EM Sample Preparation Protocol

The complex was first dialyzed against 200 ml of intermediate buffer (20 mM Hepes, 200 mM KAc, 200 mM Na-Glu, 5 mM MgAc₂, 0.5 mM TCEP, pH 7.5) for 2 h at 4 °C using Slide-A-Lyzer MINI Dialysis Units (7000MWCO) (Thermo Fischer Scientific). Then, a second dialysis was performed in 200 ml of the final cryo-EM buffer (20 mM Hepes, 100 mM KAc, 50 mM Na-glutamate, 5 mM MgAc₂, 0.5 mM TCEP, pH 7.5) for 4 h at 4 °C. Finally, 8 mM CHAPSO (Sigma-Aldrich) was added to the dialyzed complex to prevent adsorption of the particles to the air/water interface^{47 (link)}. The sample was centrifuged for 2 h at 16,000 × g to remove potential aggregates.