Cryo-EM Sample Preparation for CTPS

To prepare samples for cryo-EM, CTPS was applied to glow-discharged CFLAT 2/2 holey-carbon grids (Protochips) and blotted away four times successively, before being plunged into liquid ethane using a Vitrobot (ThermoFisher). Sample conditions for CTP-bound CTPS2 were as described previously (10 (link)). Conditions for CTP-bound CTPS1 were 5 mM CTP and 10 mM MgCl₂ in 20 mM Tris ⋅ HCl pH 7.9, 100 mM NaCl, and 0.5 mM DTT. Conditions for human and mouse CTPS bound to small molecule inhibitors were 5 μM CTPS, 2 mM UTP, 7.5 mM glutamine, 10 mM MgCl₂, and 50 μM R80 or T35 in 20 mM Tris ⋅ HCl pH 7.9, 100 mM NaCl, and 0.5 mM DTT. Data for CTP-bound CTPS1 was collected on a Glacios (ThermoFisher). Data for all other structures was collected on a Titan Krios (ThermoFisher) equipped with a Quantum Gatan Imaging Filter energy filter (Gatan Inc.) operating in zero-loss mode with a 20-eV slit width. Both microscopes were equipped with a K-2 Summit Direct Detect camera (Gatan Inc.). On the Glacios, movies were collected in counted mode with a pixel size of 1.16 Å/pixel, with 50 frames and a total dose of 65 electrons/ Ų. On the Titan Krios, movies were collected in superresolution mode with a pixel size of 0.525 Å/pixel, with 50 frames and a total dose of 90 electrons/ Ų. Data collection was automated using Leginon (61 (link)).