using the reconstructions of the two half maps as a reference and a tight soft mask focused on the LSU at a pixel size of 3.45 Å. Particles were subjected to 2–3 rounds of refining image warp grid, particle poses, stage angles, volume warp grid, defocus and pixel size. After refinements, new subtomograms and their corresponding CTF volumes were extracted at a pixel size of 6.
After refinement in M, translocon-centred OST-particles were extracted at a pixel size of 6.9 Å and subjected to classification in RELION focused on the chaperone binding site. The resulting classes were refined in M as above using masks focusing on SEC61, TRAP, OST and chaperone.Ribosomal intermediate states were obtained by hierarchical classification focused on the rotation of the SSU and on the tRNA and elongation factor binding sites.
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