Cryo-EM Sample Preparation for hDGAT1

Cryo grids were prepared using the Thermo Fisher Vitrobot Mark IV. Quantifoil R1.2/1.3 Cu grids were glow-discharged in air for 40 sec at medium level using the Plasma Cleaner (Harrick Plasma, PDC-32G-2). 3.5 μl of concentrated hDGAT1 were applied to each glow-discharged grid. After blotting with filter paper (Ted Pella, Inc. Prod.# 47000–100) for 3.5 sec, the grids were plunged into liquid ethane cooled with liquid nitrogen. Movie stacks were collected using SerialEM^{23 (link)} on a Titan Krios at 300 kV with a Quantum energy filter (Gatan) and a Cs corrector (Thermo Fisher), and at a nominal magnification of 105,000 × and defocus values of −2.0 μm to −1.2 μm. A K2 Summit direct electron detector (Gatan) was paired with the microscope. Each stack was collected in the super-resolution mode with an exposing time of 0.175 sec per frame for a total of 32 frames. The dose was about 50 e⁻/Ų for each stack. The stacks were motion corrected with MotionCor2^{24 (link)} and binned (2×2) so that the pixel size is 1.114 Å. Dose weighting^{25 (link)} was performed during motion correction, and the defocus values were estimated with Gctf^{26 (link)}.