Detector model: GATAN K2 SUMMIT (4k x 4k)
Detector mode: COUNTING
Frames per image: 1-60
Average exposure time: 9.0 s
Average electron dose per image: 0.87 e/Ų
Details: Freshly purified protein was concentrated using Vivaspin concentrating devices with a molecular weight cutoff of 100kDa to 2-2.5 mg ml-1. 2.8 ul were applied on holey-carbon cryo-EM grids (Quantifoil Au R1.2-1.3, 200 and 300 mesh), which were prior glow-discharged at 5 mA for 20 s. Grids were blotted for 3-5 s in a Vitrobot (Mark 3, Thermo Fisher) at 20C temperature and 100% humidity, subsequently plunge-frozen in liquid ethane and stored in liquid nitrogen. Cryo-EM data were collected on a 200 keV Talos Arctica microscope (Thermo Fisher) using a post-column energy filter (Gatan) in zero-loss mode, using a 20 eV slit, a 100 um objective aperture, in an automated fashion using EPU software (Thermo Fisher) on a K2 summit detector (Gatan) in counting mode. Cryo-EM images were acquired at a pixel size of 1.012A (calibrated magnification of 49,407x), a defocus range from -0.4 to 2.5 um, an exposure time of 9 sec and a sub-frame exposure time of 150 ms (60 frames), and a total electron dose on the specimen level of about 52 electrons per A2. Best regions on the grid were screened with a self-written script to calculate the ice thickness and data quality was monitored on the fly using the software FOCUS