The previous high-resolution structure of the E. coli 70S ribosome (Noeske et al., 2015 (link)) was used as a starting model. We used the ‘Fit to Map’ function in Chimera (Pettersen et al., 2004 (link)) to calibrate the magnification of the cryo-EM map of the 50S ribosomal subunit generated here to maximize correlation, resulting in a pixel size of 0.7118 Å rather than the recorded 0.71 Å. Focused-refined maps were transformed into the frame of reference of the 70S ribosome for modeling and refinement, using the ‘Fit to Map’ function in Chimera, and resampling the maps on the 70S ribosome grid. The 50S and 30S subunits were refined separately into their respective focused-refined maps using PHENIX real-space refinement (RSR; Liebschner et al., 2019 (link)). Protein and rRNA chains were visually inspected in Coot (Casañal et al., 2020 (link)) and manually adjusted where residues did not fit well into the density, making use of B-factor blurred maps where needed to interpret regions of lower resolution. Focused-refined maps on smaller regions were used to make further manual adjustments to the model, alternating with PHENIX RSR. Some parts of the 50S subunit, including H69, H34, and the tip of the A-site finger, were modeled based on the 30S subunit focused-refined map. The A-site and P-site tRNAs were modeled as follows: anticodon stem-loops, 30S subunit focused-refined map; P-site tRNA body, 50S subunit focused-refined map, with a B factor of 20 Å2 applied; A-site tRNA body, 30S subunit focused-refined map and 50S subunit focused-refined map with B factors of 20 Å2 applied; tRNA-ACCA 3’ ends, 50S subunit focused-refined map with B factors of 20–30 Å2 applied. Alignments of uS15 were generated using BLAST (Altschul et al., 1997 (link)) with the E. coli sequence as reference. The model for bL31A (E. coli gene rpmE) was manually built into the CP and 30S subunit head domain focused-refined maps before refinement in PHENIX.
A model for paromomycin was manually docked into the 30S subunit focused-refined map, followed by real-space refinement in Coot and PHENIX. Comparisons to prior paromomycin structural models (PDB codes 1J7T, 2VQE, and 4V51; Kurata et al., 2008 (link); Selmer et al., 2006 (link); Vicens and Westhof, 2001 (link)) used least-squares superposition of paromomycin in Coot. Although ring IV is in different conformations in the various paromomycin models, the least-squares superposition is dominated by rings I–III, which are in nearly identical conformations across models.
Ribosome solvation including water molecules, magnesium ions, and polyamines was modeled using a combination of PHENIX (phenix.douse) and manual inspection. The phenix.douse feature was run separately on individual focused-refined maps, and the resulting solvent models were combined into the final 30S and 50S subunit models. Due to the fact that the solvent conditions used here contained ammonium ions and no potassium, no effort was made to systematically identify monovalent ion positions. The numbers of various solvent molecules are given in .
Along with the individual maps used for model building and refinement, we have also generated a composite map of the 70S ribosome from the focused-refined maps for deposition to the PDB and EMDB for ease of use (however, experimental maps are recommended for the examination of high-resolution features). We made the composite map using the ‘Fit in Map’ and vop commands in Chimera. First, we aligned the unmasked focus-refined maps with the 70S ribosome map using the ‘Fit in Map’ tool. We then used the ‘vop resample’ command to transform these aligned maps to the 70S ribosome grid. After the resampling step, we recorded the map standard deviations as reported in the ‘Volume Mean, SD, RMS’ tool. Then, we added the maps sequentially using ‘vop add’ followed by rescaling the intermediate maps to the starting standard deviation using the ‘vop scale’ command.
A model for paromomycin was manually docked into the 30S subunit focused-refined map, followed by real-space refinement in Coot and PHENIX. Comparisons to prior paromomycin structural models (PDB codes 1J7T, 2VQE, and 4V51; Kurata et al., 2008 (link); Selmer et al., 2006 (link); Vicens and Westhof, 2001 (link)) used least-squares superposition of paromomycin in Coot. Although ring IV is in different conformations in the various paromomycin models, the least-squares superposition is dominated by rings I–III, which are in nearly identical conformations across models.
Ribosome solvation including water molecules, magnesium ions, and polyamines was modeled using a combination of PHENIX (phenix.douse) and manual inspection. The phenix.douse feature was run separately on individual focused-refined maps, and the resulting solvent models were combined into the final 30S and 50S subunit models. Due to the fact that the solvent conditions used here contained ammonium ions and no potassium, no effort was made to systematically identify monovalent ion positions. The numbers of various solvent molecules are given in .
Along with the individual maps used for model building and refinement, we have also generated a composite map of the 70S ribosome from the focused-refined maps for deposition to the PDB and EMDB for ease of use (however, experimental maps are recommended for the examination of high-resolution features). We made the composite map using the ‘Fit in Map’ and vop commands in Chimera. First, we aligned the unmasked focus-refined maps with the 70S ribosome map using the ‘Fit in Map’ tool. We then used the ‘vop resample’ command to transform these aligned maps to the 70S ribosome grid. After the resampling step, we recorded the map standard deviations as reported in the ‘Volume Mean, SD, RMS’ tool. Then, we added the maps sequentially using ‘vop add’ followed by rescaling the intermediate maps to the starting standard deviation using the ‘vop scale’ command.
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