Structural Insights into the Distortion of the Ribosomal Small Subunit at Different Magnesium Concentrations

Abstract

Magnesium ions are abundant and play indispensable functions in the ribosome. A decrease in Mg²⁺ concentration causes 70S ribosome dissociation and subsequent unfolding. Structural distortion at low Mg²⁺ concentrations has been observed in an immature pre50S, while the structural changes in mature subunits have not yet been studied. Here, we purified the 30S subunits of E. coli cells under various Mg²⁺ concentrations and analyzed their structural distortion by cryo-electron microscopy. Upon systematically interrogating the structural heterogeneity within the 1 mM Mg²⁺ dataset, we observed 30S particles with different levels of structural distortion in the decoding center, h17, and the 30S head. Our model showed that, when the Mg²⁺ concentration decreases, the decoding center distorts, starting from h44 and followed by the shifting of h18 and h27, as well as the dissociation of ribosomal protein S12. Mg²⁺ deficiency also eliminates the interactions between h17, h10, h15, and S16, resulting in the movement of h17 towards the tip of h6. More flexible structures were observed in the 30S head and platform, showing high variability in these regions. In summary, the structures resolved here showed several prominent distortion events in the decoding center and h17. The requirement for Mg²⁺ in ribosomes suggests that the conformational changes reported here are likely shared due to a lack of cellular Mg²⁺ in all domains of life.

Keywords: CryoEM; magnesium concentration; ribosome; structural distortion.

Figure 1

Subunits of the 70S ribosome were destroyed by EDTA. (a,b) E. coli cells of the BL21 DE3 (a) and MRE600 (b) strains were collected at OD₆₀₀ = 0.6 and further analyzed with a sucrose gradient in conditions with or without 10 mM EDTA. Shifting of the peaks was indicated, and the 30S and 50S peaks from the gradient with additional EDTA were collected (dashed line labeled) for cryoEM analysis. (c,d) Yeast BY4742 cells (c) and HEK293F cells (d) grown to exponential phase were collected and disrupted by a French press and homogenizer, respectively. Cell extracts were then analyzed with a sucrose gradient under conditions of 2.5 mM Mg²⁺ or 10 mM EDTA. (e,f) CryoEM images representing the typical particle shapes (white arrows) for BL21 DE3 and MRE600 (f) strains. The scale bar is labeled in white.

Figure 2

A 30S subunit at 1 mM Mg²⁺ has a flexible h44, h17, decoding center, and head. (a–c) Sucrose gradient sedimentation profile of E. coli ribosomes under 1 mM (a), 2.5 mM (b), and 10 mM (c) Mg²⁺ conditions. The 30S peak indicated in gray shadow was collected separately for cryoEM analysis. (d) Reference-free 2D classification averages for 30S particles under different conditions, as shown in (a–c). The flexible h17 and head are labeled with red arrows. (e) The overall structure and map of a 30S subunit at 1 mM Mg²⁺ concentration are represented in cartoon and surface, respectively. The rRNA helices h16/h17, h18, h27, and h44 and the S12 protein are colored in yellow, blue, green, magenta, and red. The dashed line represents the mature 30S under 10 mM Mg²⁺ conditions in this study.

Figure 3

A low Mg²⁺ concentration destabilizes the decoding center and causes a loss of S12 protein. (a–c) Three representative reconstructions were classified from 30S particles at 1 mM Mg²⁺ by applying a mask to the decoding center. A completely out-shifted h27 ((a), h27_out-3), a partially out-shifted h27 ((b), h27_out-4), and h27 in its original position ((c), h27_in-2), are represented in cartoon and surface (green) for their rigid-body-fitted structure and density map, respectively. (d,e) Two representative reconstructions with missing (d) and fully occupied S12 protein ((e), red), were classified from 30S particles at 1 mM Mg²⁺ by applying a mask to S12, as shown in the cartoon and on the surface. The rRNA helices h3 (limon), h16/h17 (yellow), h18 (blue), and h24/h45 (magenta) are also labeled according to the positions of the decoding center. Details of the interactions and movement (black arrows) of h27 and S12 are represented as inserted subfigures. A dashed line represents the mature 30S under 10 mM Mg²⁺ conditions in this study.

Figure 4

The h17 helix shifted outwards to the tip of h6. (a) Interactions between h17 (red), h10 (green), and h15 (blue) in the reconstruction of h17_in. The direction of h17 is labeled with a black arrow. (b) Interactions between h17 and S16 proteins in the reconstruction of h17_in. (c) Shifting of h17 from h17_in to h17_out. The shifted angle was measured in Chimerax, and the direction of h17 in the h17_out reconstruction is labeled with a black arrow. (d) The interactions between the out-shifted h17 and the tip of h6.

Figure 5

The movements of the different structural blocks are correlated. (a) Occupancy analysis of 30S_1mM particles displayed as a heatmap. Rows (500) correspond to sampled density maps, and columns (32) correspond to structural elements defined by the atomic model. Only the 30S body was analyzed. (b) Atomic models of the 30S subunit used for subunit occupancy analysis are colored according to the structural blocks defined through hierarchical clustering in (a). Structural features of interest are annotated. (c) Correlation between h17 and h27 in the reconstructions classified with different masks. The mask on h17 defined four different states of h17, and the mask on the decoding center defined six states of h27. Particles in each state were selected, and the distribution of particles in different h17 conformations was calculated. For each h27 state, the difference in distribution compared to the total particles is colored in red. (d) Same as (c), but the correlation between h17 and S12 was calculated.

Figure 6

The effect of Mg²⁺ concentration on the assembly of 70S. (a–d) Sucrose gradient profiles for crude 70S at 0.5 mM (a), 5 mM (b), and 20 mM (c) Mg²⁺ concentrations. (d) Profile of 30S and 50S purified from 1 mM Mg²⁺ in a gradient containing 10 mM Mg²⁺. Fractions corresponding to 30S and 50S under 1 mM Mg²⁺ conditions (Figure 2a) were pooled, and sucrose was removed, followed by reloading onto a sucrose gradient containing 10 mM Mg²⁺.

Figure 7

A model of 30S structural distortion at a low Mg²⁺ concentration. Here, 2.5 mM Mg²⁺ first causes the destabilization of h44 and partial loosening of h17, and the 30S head becomes more flexible. A further decrease in Mg²⁺ to 1 mM shifts h17 toward the tip of h6, and h16, h18, and h27 become flexible at the same time. Ribosomal S12 near the decoding center starts to leave, causing an irreversible 30S if no additional protein is supplied. EDTA incubation was used to extract all the Mg²⁺ from ribosomes and completely destroy the 30S subunit, release the ribosomal proteins, and linearize the 16S rRNA.