(A) Cutaway view through the local resolution map of the 70S ribosome reconstruction. (B) Base pair density in the cores of the 30S (left) and 50S (right) ribosomal subunits. Examples demonstrate …
The number of particles at each stage is shown, as well as the final resolutions as defined by half-map FSCs (cutoff of 0.143).
(A) Half-map FSC curve for the 70S ribosome is shown. The ‘gold-standard’ cutoff value for resolution (0.143) is indicated. (B) The map-to-model FSC curve for the 70S ribosome, with an overall …
(A) Local resolution of the 30S and 50S subunits. Brackets indicate regions masked for further focused refinement. 16S rRNA helix h44 and 23S rRNA helices H34 and H69 are also labeled. (B) Half-map …
Examples include magnesium ions with different levels of direct coordination to the rRNA or proteins. The fully-hydrated Mg2+ is located near 23S rRNA nucleotide A973.
(A) Post-transcriptionally modified nucleotides and post-translationally modified amino acids in the 30S subunit. The density for m7G527 and mSAsp89 is contoured at a lower level to show the …
Both m7G527 (light purple) and β-methylthio-Asp89 in uS12 (pink) appear hypomodified based on the cryo-EM map contour level required to enclose adjacent atoms and residues.
(A) Map around nucleotides C2443, C2442, and U2441 of the 50S subunit in a reconstruction from frames 1–2 of the exposure, corresponding to the first ~2 electrons per Å2. The same nucleotides are …
(A) Overall view of P-site tRNA anticodon stem-loop (ASL, cyan), mRNA (red), 16S rRNA nucleotides (light purple), and uS9 residues (gold). (B) Interactions between 30S subunit head nucleotide G1338 …
(A) Solvation of the 30S subunit with solvent oxygen atoms shown as red spheres. (B) Polyamine (gray carbon) and Mg2+ (green) sites in the 30S subunit, shown with metal-coordinating atoms (oxygen in …
(A) bS21 C-terminal structure in the 30S subunit head-focused map, with Shine-Dalgarno helical density shown in gray and density for bS21 in rose. Low-pass filtering to 3.5 Å resolution was applied …
Sequence alignments of phage S21 homologs (in red) and associated host bacterial clades from (A) Bacteroidetes (orange), (B) The Candidate Phyla Radiation (magenta), (C) Betaproteobacteria (green) …
(A) Model of isoAsp at residue position 119 in uS11, with nearby residues and cryo-EM density from the 30S subunit platform-focused refinement. Weak density for the carboxylate is consistent with …
Frames 1–3 of the acquired movies were used to calculate a 30S focused-refined cryo-EM map. (A) The model of isoaspartate 119 in uS11 and neighboring amino acids is shown in the density. The arrow …
(A) Phylogenetic tree of the uS11 ribosomal proteins in eukaryotes, including both cytoplasmic and organelle examples, along with Escherichia coli and the amino acids around the PHNG motif. The …
Comparisons of published uS11 models (blue) in the archaeal (Nürenberg-Goloub et al., 2020) (A) and eukaryotic (Tesina et al., 2020) ribosomes (B) with models incorporating the IsoAsp modification …
(A) Chemical structure of paromomycin (PAR) with ring numbering. (B) Comparison of paromomycin conformations in different structures. Paromomycin from three prior structural models (Kurata et al., …
(A) Interaction of the C-terminus of uS15 (light blue) with 23S rRNA nucleotides 713–715 (purple). The 30S subunit cryo-EM map is shown with a B factor of 20 Å2 applied. (B) Interaction between uS13 …
Comparison of solvation in the PTC near E. coli nucleotide G2447 to that in phylogenetically diverse 50S subunits. Solvent molecules conserved in bacterial ribosomes from E. coli, S. aureus, and T. …
(A) The map-to-model FSC curves calculated with the map from Stojković et al., 2020 (EMD-20353) against the associated model (orange; PDB: 6PJ6) and the 50S model presented here (blue). (B) The map-t…
(A) Water molecules modeled in the 50S subunit are shown as red spheres (oxygen atoms) in the outline of the map. The view is from the 30S subunit interface side, with approximate locations of uL1 …
(A) Structural model of thioamide between Met82 and Gly83 in uL16 (mint), with the 50S subunit cryo-EM density map contoured at two levels to highlight sulfur and phosphorus atoms. The lower contour …
(A) Structural model of thioamide between Met82 and Gly83 in uL16 (mint), with the cryo-EM density of map EMD-20353 contoured at two levels to highlight sulfur and phosphorus atoms. The lower …
Magnification | 109,160 |
Voltage (kV) | 300 |
Spherical aberration (mm) | 2.7 |
Electron exposure (e–/Å2) | 39.89 |
Defocus range (μm) | −0.6/−1.5 |
Pixel size (Å) | 0.7118 |
Symmetry imposed | C1 |
Initial particle images (no.) | 874,943 |
Final particle images (no.) | 307,495 |
Map resolution (Å) | 2.02 |
Map resolution with Ewald correction (Å) | 1.98 |
FSC threshold (gold-standard) | 0.143 |
Model | Without Ewald correction (Å) | Ewald sphere corrected (Å) | Map sharpening B factor for Ewald (Å2) |
---|---|---|---|
30S subunit | 2.15 | 2.11 | −25.7 |
30S subunit head domain | 2.09 | 2.01 | −19.7 |
30S subunit platform | 2.12 | 2.08 | −21.8 |
50S subunit | 1.92 | 1.9 | −25.1 |
50S subunitcentral protuberance | 2.28 | 2.26 | −21.5 |
70S ribosome | 2.06 | 2.04 | −29.5 |
* Map-vs-model FSC with threshold = 0.5.
Model component | 70S ribosome | 30S subunit | 50S subunit |
---|---|---|---|
Model resolution, Ewald-corrected map (Å) | 2.04 | 2.11 | 1.9 |
FSC threshold (map-vs.-model) | 0.5 | 0.5 | 0.5 |
Map sharpening B factor (Å2) | −29.5 | −25.7 | −25.1 |
Model composition | |||
non-hydrogen atoms | 149356 | 54550 | 91592 |
Mg2+ ions | 309 | 93 | 218 |
Zn2+ ions | 2 | 0 | 2 |
polyamines | 17 | 2 | 15 |
waters | 7248 | 2413 | 4835 |
ligands (paromomycin) | 1 | 1 | 0 |
B factors (Å2) | |||
RNA | 23.83 | 28.09 | 20.9 |
protein | 24.42 | 28.91 | 20.95 |
waters | 20.66 | 17.94 | 22.02 |
other | 29.29 | 20.35 | 33.12 |
R.m.s. deviations from ideal values | |||
Bond (Å) | 0.006 | 0.005 | 0.006 |
Angle (°) | 0.952 | 0.838 | 0.997 |
Molprobity all-atom clash score | 7.34 | 7.12 | 7.02 |
Ramachandran plot | |||
Favored (%) | 96 | 95.66 | 96.26 |
Allowed (%) | 3.87 | 4.17 | 3.65 |
Outliers (%) | 0.13 | 0.17 | 0.1 |
RNA validation | |||
Angles outliers (%) | 0.02 | 0.009 | 0.02 |
Sugar pucker outliers (%) | 0.46 | 0.39 | 0.39 |
Average suiteness | 0.579 | 0.586 | 0.583 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (species) | Escherichia coli (MRE600) | Gift of Arto Pulk, UC Berkeley | ATCC #29417, NCTC #8164 | Strain with low ribonuclease activity |
Other | 300 mesh R1.2/1.3 UltraAuFoil grids | Electron Microscopy Sciences | Q350AR13A | |
Software, algorithm | SerialEM | Schorb et al., 2019 | RRID:SCR_017293 | |
Software, algorithm | MotionCor2 | Zheng et al., 2017 | RRID:SCR_016499 | |
Software, algorithm | CTFFind4 | Rohou and Grigorieff, 2015 | RRID:SCR_016732 | |
Software, algorithm | RELION | Zivanov et al., 2018 | Version 3 and 3.1 RRID:SCR_016274 | |
Software, algorithm | Cryosparc | Punjani et al., 2017 | Version 2 RRID:SCR_016501 | |
Software, algorithm | Chimera | Pettersen et al., 2004 | RRID:SCR_004097 | |
Software, algorithm | PHENIX | Liebschner et al., 2019 | RRID:SCR_014224 | |
Software, algorithm | Coot | Casañal et al., 2020 | RRID:SCR_014222 |
Phages encoding S21 homologs.
Tabs include phages encoding S21 homologs with predicted bacterial hosts, along with ribosome binding sites for the phages, Betaproteobacteria, Firmicutes, CPR bacteria, Spirochaetes, and Bacteroidetes.
Phylogenetic analysis of rRNA contacts near the uS11 isoAsp residue.
Tabs include 16S base pair statistics for prokaryotes, bacteria, archaea, 16S rRNA genome information for prokaryotes, 18S base pair statistics for eukaryotes, 18S rRNA genome information for eukaryotes, and nucleotide statistics for position 718. All 16S rRNA base pairs and position 718 are with E. coli numbering. 18S rRNA base pairs are with S. cerevisiae numbering.