Cryo-EM Sample Preparation Protocol

A volume of 3 µL of purified ADH (~2.5 mg mL⁻¹), metHb (~12 mg mL⁻¹), or inhibited PKA (~5 mg mL⁻¹) were dispensed on UltrAuFoil R1.2/1.3 300-mesh grids (Electron Microscopy Services) that had been freshly plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.) with a 75% argon/25% oxygen atmosphere at 15 Watts for 6 s. Grids were blotted manually^{29 (link)} using a custom-built manual plunger in a cold room (≥95% relative humidity, 4 °C)^{12 (link)}. Samples were blotted for 4–5 s with Whatman No. 1 filter paper immediately before plunge freezing in liquid ethane cooled by liquid nitrogen. We aimed to achieve a particle concentration that maximized the number of particles contained within the holes without resulting in overlapping particles and/or aggregation in order to maintain a consistent ice thickness across the center of holes and provide sufficient signal for accurate CTF estimation, as observed previously^{12 (link)}.