Frozen-hydrated specimens were imaged at −170 °C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a direct detection device (Gatan K2 Summit). The microscope was operated at 300 kV with a magnification of ×9,400, resulting in an effective pixel size of 4.5 Å at the specimen level. We used SerialEM (38 (link)) to collect low-dose, single-axis tilt series with dose fractionation mode at about 8 µm defocus. Tilt series were collected from −51° to 51° with an increment of 3° and a cumulative dose of ∼70 e−/Å2 distributed over 35 stacks. Each stack contains approximately eight images. We used Tomoauto (39 ) to facilitate data processing, which includes drift correction of dose-fractionated data using Motioncorr (40 (link)) and assembly of corrected sums into tilt series, automatic fiducial seed model generation, alignment, and contrast transfer function correction of tilt series by IMOD (41 (link)) and weighted back projection (WBP) reconstruction of tilt series into tomograms using Tomo3D (42 (link)). Each tomographic reconstruction is 3,710 × 3,838 × 1,600 pixels and ∼1.30 Gb in size. In total, 4,227 tomographic reconstructions from six different strains were generated.