Quantifoil Cu R1.2/1.3 grids (Quantifoil Micro Tools GmbH, Germany) were washed in chloroform and air-dried. Aliquots of 4-μl samples at a concentration of approximately 5 mg/ml were loaded onto the grids, which were glow-discharged as routine. Grids were blotted for 2.5 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using a FEI Vitrobot IV (FEI Company). Grids were checked in FEI Tecnai T12 operated at 120 kV to ensure that the thickness of ice was proper for data collection and that there was no obvious preferred orientation observed. Grids were transferred to Titan Krios operated at 300 kV for data collection. Images were recorded on a Gatan K2 Summit direct electron detector operated in super-resolution counting mode following the established dose fractionation data acquisition protocol (38). Images were recorded at a nominal magnification of ×22,500, corresponding to a calibrated super-resolution pixel size of 0.66 Å on the specimen. The dose rate on the detector was set to be ~8.2 counts per physical pixel per second. The total exposure time was 8 s, leading to a total accumulated dose of 50 electrons/Å2 on the specimen. Each image was fractionated into 32 frames, each with an accumulation time of 0.25 s. Dose-fractionated images were recorded using a semiautomated acquisition program UCSF Image4 (written by X. Li). Defocus values ranged from −0.8 to −2.5 μm.

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