Sample vitrification and cryo-EM data acquisition

Glow-discharge the grids at 25 mA for 30 sec with PELCO easiGlow system (Ted Pella)

Apply 3 μl of protein solution to the grid:

QUANTIFOIL R1.2/1.3 mesh 300 (Electron Microscopy Sciences), or

QUANTIFOIL R2/1 mesh 300 (Electron Microscopy Sciences)

Vitrify the sample with a Vitrobot cryo-plunger (Thermo Fisher Scientific)

(Optional) Add 0.05%(w/v) of n-Octyl-Beta-D- Glucopyranoside as a surfactant before vitrification.

Collect dataset at a magnification of 81,000x and a corresponding pixel size of 1.05 Å and a defocus range of -0.8 to -2.0 μm. Image stacks contain 50 frames with a total dose of 50 e/Å2.

Collect dataset at a magnification of 36,000x and a corresponding pixel size of 0.5575 Å and a defocus range of -0.8 to -2.0 μm in a super-resolution correlated-double sampling mode. Image stacks contain 50 frames with a total dose of 50 e/Å2.