Paromomycin

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 Ų applied; A-site tRNA body, 30S subunit focused-refined map and 50S subunit focused-refined map with B factors of 20 Ų applied; tRNA-ACCA 3’ ends, 50S subunit focused-refined map with B factors of 20–30 Ų 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.

Calcofluor White Stain, a premixed CF (Fluorescent Brightener 28) and EB dye, was purchased from Sigma-Aldrich (St. Louis, Mo, USA). CF and EB were from Sigma-Aldrich and Nacalai Tesque (Kyoto, Japan), respectively. N-Lauroylsarcosine sodium salt (>94.0% purity) was from Sigma-Aldrich.
Lactacystin (>94.2% purity) and polyoxin D (>94.5% purity) were purchased from Biolinks Co. Ltd. (Tokyo, Japan) and Kaken Pharmaceutical Co. Ltd. (Tokyo, Japan), respectively, whereas metronidazole (>98.0% purity) and paromomycin (>98.0% purity) were both from Sigma-Aldrich. All four compounds were dissolved in sterilized water, respectively, at 1, 10, 50, and 50 mM as the stock solutions. Aliquots of 50 μL were stored at −30°C, and freeze-thaw cycles were less than two before use.
The Pathogen Box, 400 compounds exhibiting diverse scaffolds, was provided by MMV (https://www.pathogenbox.org/); each compound was dissolved in DMSO at 10 mM and distributed into individual wells of 96-well plates (10 μL/compound and 80 compounds/plate). Ninety microliters of DMSO were then added to each well and then 10 replicates were made (10 μL aliquots of all the compounds' stocks at 1 mM) and stored at −30°C. When needed, a set of replicates covering all 400 compounds (10 μL aliquoted at 1 mM each) was thawed, and 1 μL for the trophozoite proliferation assay and 2.4 μL for the cyst formation assay was dispensed into wells of a 96-well culture plate to make a replicate. Auranofin (>98% purity) (which is identified as E-H-05 in the Pathogen Box) was also purchased from Sigma-Aldrich, dissolved in DMSO to 10 mM and dispensed into 50 μL aliquots for storage at −30°C.

To assemble 70S ribosomes, 0.5 mg of U2554C U2555C (CC) mutant 50S subunit and 0.6 mg of WT untagged 30S subunit were incubated in buffer C with 10 mM MgCl₂ at 37°C for 45 min. The ribosome mixture was then loaded onto a 15–40% (w/v) sucrose gradient in buffer C with 10 mM MgCl₂. Gradients were centrifuged at 28 000 rpm (97 000 × g) for 16 h in a SW-32 rotor (Beckman-Coulter). An ISCO gradient fraction system was used to isolate the 70S fraction (Supplementary Figure S2).
Ribosome–tRNA–mRNA complexes were prepared as previously described (35 (link)) with modifications. Complexes were formed non-enzymatically by combining 2 μM Met-tRNA^fMet, 5 μM mRNA, and 100 nM ribosomes in buffer AC (20 mM Tris pH 7.5, 100 mM NH₄Cl, 15 mM MgCl₂, 0.5 mM EDTA, 2 mM DTT, 2 mM spermidine and 0.05 mM spermine). Paromomycin at a concentration of 100 μM was used to ensure nonenzymatic A-site tRNA binding. The complex was incubated at 37°C for 30 min and then held at 4°C. The mRNA sequence was 5′-GUAUAAGGAGGUAAAAUGAUGUAACUA-3′ (IDT). Met codons are underlined. This mRNA sequence placed Met-tRNA^fMet in both the A and P sites. Cryo-EM grids were prepared and samples were frozen as previously described (35 (link)).

Excysted sporozoites were cotransfected with 50 μg repair template and 30 μg CRISPR/Cas9 targeting plasmid, as previously described (24 (link), 29 (link)). Electroporated sporozoites were transferred to cold DPBS and stored on ice until infection. To select for transgenic parasites, three GKO mice per transgenic parasite line were administered 8% (wt/vol) sodium bicarbonate by oral gavage for 5 min prior to infection and then subsequently administered 2.5 × 10⁷ electroporated sporozoites in DPBS by oral gavage. Paromomycin (16 g/L; Biosynth International, Inc.) was added to drinking water 24 hpi. Fecal pellets were collected at 3-day intervals and stored at −80°C for DNA extraction or at 4°C for luciferase assays or for subsequent infections. Following confirmation of proper template integration, a fecal slurry containing 2 × 10⁴ oocysts from an infected GKO mouse was used to infect three to six NSG mice, as described previously (29 (link)). Paromomycin (16 g/L; Biosynth International, Inc.) drinking water was offered continuously beginning immediately after infection, and fecal pellets were isolated and tested as described above.