# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 2 Results
### Determination of the Structure of 21S  
We characterized the structure of purified 21S particles with cryo-EM. After semi-manual picking with CryoSPARC, we selected 172,000 particles. Following 3D classification, we retained a main class of 49,000 particles, with 28% of the particles classified (Supplementary Figure S3). This allowed us to produce a final 3D reconstruction at a resolution  
Figure 4: Relative quantification of 21S proteins by quantitative mass spectrometry. (**A**) Histogram of the ratio between purified 21S and 30S ribosomal particles analyzed by LC-MS/MS. Protein levels are normalized against those of the bS20 proteins used to purify the particles and are the means of three biological replicates, with standard deviation indicated. (**B**) Schematic representation of the proportion of ribosomal proteins in the Nomura assembly map, adapted from [7]. The ratio of proteins in 21S is orange, while the proteins from 30S are normalized to 1 and shown in blue. of 6.1 A (Figure 5A), which was detailed enough for us to describe large conformational changes and explore protein occupancy levels. The 21S head is slightly more rotated than in a mature 30S (Figure 5B,C). Contrary to other immature 30S subunits that accumulate in \(\Delta rimM\) cells [12], the entire helix 44 is clearly visible in the 21S, with its upper region latched onto the decoding center (Figure 5D). To estimate the protein occupancy of our 21S particles, we compared our reconstruction with a model of the _E. coli_ 30S ribosomal subunit (PDB 4V4Q) filtered at the same resolution. Proteins uS2, bS21, and bS1 are clearly missing from our 21S reconstruction, and the uS7 protein is very poorly defined (Figure 6A). For the most part, these observations match the occupancy levels observed using mass spectrometry. Indeed, the 21S missing proteins uS2, bS21, and bS1 are the least present in the proteomics quantification experiments (Figure 4A, group 3). The notable exception is uS7, which is relatively abundant in our quantification results (Figure 4A, group 2), but whose low cryo-EM density suggests a partial occupancy in 21S. This primary protein binds early to the 3\({}^{\prime}\) domain of the 16S rRNA to initiate the 30S assembly, resulting in the fixation of seven other ribosomal proteins (Figure 4B). While the lack of uS7 and not its related secondary proteins in our 21S structure would seem to contradict a strictly sequential and cooperative ribosome assembly scheme, it was recently shown that uS7 binding dynamics during assembly gradually decrease as additional components are installed [31]. The results from [PERSON] et al. suggest a different and more flexible process than previously thought, with the later-binding ribosomal proteins also chaperoning the rRNA folding early in assembly, as well as regulating the dynamics of protein-RNA binding during the 30S assembly. The differences observed could therefore be explained by the different methods employed [29]. Indeed, MS analysis reflects the average protein occupancy of the entire population, while cryo-EM structures are created from individual populations selected in silico during the single-particle refinement process. In addition, some proteins (such as uS7) may appear in the cryo-EM map because of their low flexibility or high affinity. The two methods are, however, complimentary, and their combination provides both general and individual details about the particles being observed. In fact, our structure's global conformational changes and missing ribosomal proteins are very similar to what happens when mature 30S subunits encounter RsgA (also known as YjeQ). Although this ribosome assembly factor was absent in our reconstruction (Figure 6B), it intervenes during the later stages of 30S maturation [32; 33], along with Era, RbfA, and KsgA. RsgA binds to immature  
Figure 5: Cryo-EM structure of the 21S particle. (**A**) The map is colored to reflect the local resolutions computed with ResMap.