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Micrographs collected using a Thermo Fisher Krios 300 kV electron microscope equipped with a Falcon 4 camera and Selectris X energy filter at a nominal magnification of 81,000× (pixel size of 0.936 Angstrom) and a 100 µm objective aperture. Movies were collected to a total fluence of 50 electrons per angstom squared with 1800 frames in EER mode targeting a defocus of 0.5 µm. Cytoplasm was targeted by selecting regions of interest in a low-magnification overview.
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Two-dimensional template matching result files from the leopard em program match template where the file patterns follow the base name of the reference micrograph plus the result type. The file “*output_mip.mrc” Corresponds to the maximum intensity projection over the entire search space. The files “*output_phi.mrc”, “*output_theta.mrc”, “*output_psi.mrc”, and “*output_relative_defocus” correspond to the orientations, in ZYZ Euler angle format, and defocus offset from the micrograph which produced the maximum intensity projection. The files “*output_correlation_average.mrc” and “*output_correlation_variance.mrc” correspond to the mean and variance of the cross-correlation value over the entire search space. The files “*output_scaled_mip.mrc” are the maximum intensity projection values scaled to be z-scores over the whole search space.
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Two-dimensional template matching result files from the leopard em program match template where the file patterns follow the base name of the reference micrograph plus the result type. The file “*output_mip.mrc” Corresponds to the maximum intensity projection over the entire search space. The files “*output_phi.mrc”, “*output_theta.mrc”, “*output_psi.mrc”, and “*output_relative_defocus” correspond to the orientations, in ZYZ Euler angle format, and defocus offset from the micrograph which produced the maximum intensity projection. The files “*output_correlation_average.mrc” and “*output_correlation_variance.mrc” correspond to the mean and variance of the cross-correlation value over the entire search space. The files “*output_scaled_mip.mrc” are the maximum intensity projection values scaled to be z-scores over the whole search space.
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Two-dimensional template matching result files from the leopard em program match template where the file patterns follow the base name of the reference micrograph plus the result type. The file “*output_mip.mrc” Corresponds to the maximum intensity projection over the entire search space. The files “*output_phi.mrc”, “*output_theta.mrc”, “*output_psi.mrc”, and “*output_relative_defocus” correspond to the orientations, in ZYZ Euler angle format, and defocus offset from the micrograph which produced the maximum intensity projection. The files “*output_correlation_average.mrc” and “*output_correlation_variance.mrc” correspond to the mean and variance of the cross-correlation value over the entire search space. The files “*output_scaled_mip.mrc” are the maximum intensity projection values scaled to be z-scores over the whole search space.
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60S_map_px0.936_bscale0.5.mrc corresponds to the 60S ribosome template simulated at 0.936 Angstroms per pixel with a B-factor scaling of 0.5 applied to the deposited PDB per-atom B-factors. 60S_map_px0.95_bscale0.5.mrc is the same, except simulated at 0.95 Angstroms per pixel. SSU-body_map_px0.936_bscale0.5.mrc is the simulated volume of the 40S ribosome body template (no head) at 0.936 Angstroms per pixel. All simulations were done with the Python package ttsim3D.
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The particle positions from the Leopard-EM results files were used to extract the particles in 512 by 512 pixel boxes, which were then normalized to have a mean of zero and a standard deviation of one. These extracted particles were saved as .mrcs files, with one file generated per micrograph. Subsequently, RELION compatible STAR files were created using the Euler angles and CTF parameters in the Leopard-EM results. 3D volumes were generated for all particles, non-rotated particles, and rotated particles, using RELION's reconstruct program. After low-pass filtering the maps to 30 Angstrom, masks for the full 80S ribosome were generated in RELION. These masks were manually modified in ChimeraX to create a mask for the SSU body. Postproccesing was performed in RELION with an automatically determined B-factor to generate the masked 3D maps and estimate the resolution.
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