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Abstract
Cryo-electron microscopy is now used as a method of choice in structural biology for studying protein synthesis, a process mediated by the ribosome machinery. In order to achieve high-resolution structures using this approach, one needs to obtain homogeneous and stable samples, which requires optimization of ribosome purification in a species-dependent manner. This is especially critical for the bacterial small ribosomal subunit that tends to be unstable in the absence of ligands. Here, we report a protocol for purification of stable 30 S from the Gram-positive bacterium Staphylococcus aureus and its cryo-EM structures: in presence of spermidine at a resolution ranging between 3.4 and 3.6 Å and in its absence at 5.3 Å. Using biochemical characterization and cryo-EM, we demonstrate the importance of spermidine for stabilization of the 30 S via preserving favorable conformation of the helix 44.
Highlights
Protein synthesis is a tightly regulated biological process performed by the ribosome
The shift of the peak towards light fractions of the gradient and the peak asymmetry suggested the loss of structural integrity of these subunits (Supplementary Figure S1B)
To further monitor the integrity of the dissociated ribosomal subunits, the 50 S and 30 S peaks were loaded onto sucrose gradients equilibrated in a buffer containing 200 mM KCl
Summary
Protein synthesis is a tightly regulated biological process performed by the ribosome. The bacterial ribosome (70 S) can be divided into the large (50 S) and the small (30 S) subunits The latter, which contains the decoding center, consists of 21 proteins (r-proteins) and of the 16 S RNA (rRNA). Its structure needs to be stable enough to be maintained during translation elongation and sufficiently flexible to allow mRNA and factors binding during translation initiation For these reasons, several proteins (e.g., Era, RbfA, RimM), which are involved in the ribosome biogenesis, perform quality control function of the h44 as one of the last checkpoints of the 16S rRNA maturation (Dammel and Noller, 1995; Bylund et al, 1998; Datta et al, 2007; Guo et al, 2013; Razi et al, 2019; Schedlbauer et al, 2020). For efficient participation of the 30 S in protein synthesis, flexibility and structural integrity are required at the same time, and especially proper folding of h44 is essential
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