Fei vitrobot mark 4 system

To prepare the cryo-EM sample for structure determination, 2–3 µL of purified DGAT1 in PMAL-C8 with 5 µM inhibitor was applied to Quantifoil holey carbon grid (Cu R1.2/1.3; 400 mesh) glow discharged for 30 s. Optimal particle distribution was obtained with a protein concentration of 4–5 mg/mL. The grids were blotted with a Whatman #1 filter paper for 5 s with ~95% humidity at 4 °C and plunged frozen in liquid ethane using an FEI Vitrobot Mark IV system (Thermo Fisher Scientific). Cryo-EM data were collected on a Talos Arctica or a Titan Krios electron microscope (Thermo Fisher Scientific). Images were recorded using SerialEM^{43 (link)}. Refer to Supplementary Table 1 for detailed information about microscope type and data collection parameters.

Purified DNA-PKcs, Ku70/80 and 43-bp DNA were mixed at a molar ratio of 1.2:1:1 in 20 mM HEPES pH 7.6, 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl₂, 2 mM DTT and 1 mM M3814 and incubated on ice for 1 h. After incubation, 3 μl of mixture was directly loaded to a Quantifoil R1.2/1.3 grid 300 Mesh Cu with graphene oxide (GO). Protein samples were left on a grid for 20 s, then blotted and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV system (Thermo Fisher Scientific) at 4 °C and 100% humidity. The grids were then clipped and transferred to a Titan Krios electron microscope operating at a voltage of 300 kV with a K3 direct electron detector (Gatan) at the cryo-EM facility of the Department of Biochemistry, University of Cambridge. Automated data collection was performed using the Thermo-Scientific EPU software package (Table 1).

DNA-PKcs was incubated on ice for 1 h with 1 mM target ligand (ATPγS, wortmannin, NU7441, AZD7648 or M3814) and then loaded onto a Quantifoil R1.2/1.3 grid with graphene oxide. For the homemade single-layer graphene oxide, the grids were previously glow discharged for 120 s at a current of 15 mA and the graphene oxide preparation was based on the previous protocol of Bokori-Brown et al.^{35 (link)}. Protein samples were left to adhere for 20 s and later blotted and plunge-frozen in liquid ethane using the FEI Vitrobot Mark IV system (ThermoFisher Scientific) at 4 °C and 100% humidity. The grids were transferred to a Titan Krios electron microscope operating at a voltage of 300 kV with a K3 direct electron detector (Gatan) at the cryo-EM facility of the Department of Biochemistry, University of Cambridge. All datasets were collected in super-resolution counting mode with a magnification of ×130,000.