Structure of the bacterial ribosome at 2 Å resolution
- Department of Chemistry, University of California, Berkeley, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Innovative Genomics Institute, University of California, Berkeley, United States
- Earth and Planetary Science, University of California, Berkeley, United States
- Department of Chemistry, Yale University, United States
- Environmental Science, Policy and Management, University of California Berkeley, United States
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 7 supplements
Overall structure of the 70S ribosome and cryo-EM map quality.
(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 …
Figure 1—figure supplement 1
General scheme of cryo-EM data processing workflow.
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).
Figure 1—figure supplement 2
Fourier shell correlations for cryo-EM maps of the 70S ribosome.
(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 …
Figure 1—figure supplement 3
Resolution of maps of the 30S and 50S ribosomal subunits.
(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 …
Figure 1—figure supplement 4
Gallery of Mg2+ coordination states observed in the 50S subunit.
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.
Figure 1—figure supplement 5
Gallery of post-transcriptionally modified nucleotides and post-translationally modified amino acids.
(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 …
Figure 1—figure supplement 6
Close proximity of m7G527 in 16S rRNA and β-methylthio-Asp89 in uS12.
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.
Figure 1—figure supplement 7
Weak RNA backbone density in the 50S subunit.
(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 …
Figure 2 with 1 supplement
tRNA binding to the 30S ribosomal subunit.
(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 …
Figure 2—figure supplement 1
Solvation of the 30S ribosomal subunit.
(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 …
Figure 3 with 1 supplement
Protein bS21 interactions with the 30S ribosomal subunit head domain.
(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 …
Figure 3—figure supplement 1
Phage S21 and bacterial host bS21 sequence alignments.
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) …
Figure 4 with 3 supplements
Isoaspartyl residue in protein uS11.
(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 …
Figure 4—figure supplement 1
Cryo-EM density for uS11 based on early movie frames.
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 …
Figure 4—figure supplement 2
Conservation of residues near the isoAsp residue in uS11 homologs.
(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 …
Figure 4—figure supplement 3
Structural models for isoaspartate in archaeal and eukaryotic ribosomes.
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 …
Figure 5
Binding of paromomycin to the mRNA decoding site in the 30S subunit.
(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., …
Figure 6
Peripheral contacts between the 30S and 50S subunits.
(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 …
Figure 7 with 2 supplements
Conserved solvation in the PTC in the 50S ribosomal subunit.
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. …
Figure 7—figure supplement 1
The map-to-model resolution estimates for deposited 50S subunit structures.
(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…
Figure 7—figure supplement 2
Solvation in the 50S ribosomal subunit.
(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 …
Figure 8 with 2 supplements
Thioamide modification in protein uL16.
(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 …
Figure 8—figure supplement 1
Thioamide density in other E. coli 50S ribosomal subunit cryo-EM reconstructions.
(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 …
Figure 8—figure supplement 2
Phylogeny of YcaO family members.
(A) Phylogenetic tree of YcaO family members. The maximum likelihood tree was constructed under an LG+F+G4 model of evolution. Scale bar indicates the average substitutions per site. The inner …
Tables
Table 1
Data collection and processing.
| 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 |
Table 2
Model resolutions for subunits and domains.
| 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 |
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* Map-vs-model FSC with threshold = 0.5.
Table 3
Model refinement statistics.
| 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 |
Key resources table
| 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 |
Additional files
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Supplementary file 1
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.
- https://cdn.elifesciences.org/articles/60482/elife-60482-supp1-v2.xlsx
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Supplementary file 2
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.
- https://cdn.elifesciences.org/articles/60482/elife-60482-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60482/elife-60482-transrepform-v2.docx
