CryoET Toolbox (https://github.com/ffyr2w/cet_toolbox) was used for on-the-fly pre-processing of K2 datasets (freely available). In summary, movie frames were Fourier-cropped to a final pixel size of 1.35 Å and motion corrected by averaging frames for each tilt using program ‘alignframes’ implemented in the IMOD package52 or MotionCor253. For the Falcon4 data, 30 EER frames were grouped to create 7 dose fractions from 210 frames (last 7 frames were discarded) and motion corrected using Relion3.154. Tilt series were aligned using the default parameters in IMOD version 4.10.22 with the eTomo interface52, using gold-fiducial markers. The alignment parameters including the projection transformations, local alignments, and fitted tilt angles were then passed to emClarity32 framework. Sub-tomogram alignment and averaging were carried out in emClarity following the published protocol32. Briefly, the workflow was as follows: Particles were picked from 6× binned non-CTF-corrected tomograms by emClarity template matching function using EMD-840333 low-pass filtered to 25 Å as template. The template matching results were cleaned automatically on basis of geometrical restraints using ‘removeNeighbours’ function implemented in emClarity. Only particles that had at least three neighbours within 100 Å and oriented within 20° were retained. Subtomograms at the air-water interface were manually discarded using IMOD52. Following template matching, the data set was randomly split into two groups based on tomogram of origin, rather than randomly by sub-tomograms, which were processed independently for all subsequent steps. The initial positions and orientations of the first cycle averaging come from template matching results. C6 symmetry was applied throughout all sub-tomogram averaging procedures. The 3D alignment procedures were carried out gradually with binning of 5, 4, 3, 2 and 1. At each binning, duplicate particles were removed and the tilt-series geometry was refined using the positions of subtomograms as fiducial markers (TomoCPR). The Fourier Shell Correlation was calculated by the gold-standard method from even and odd data sets. Density maps were visualized in Chimera55 or PyMol (Schrödinger, Inc.). A diagram for the data processing is presented in Supplementary Fig. 3.

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