# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 4 Materials and Methods
### Protein Identification Using Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS)
#### 4.7.3 Cryo-Electron Microscopy  
After diluting 150 nM ribosomes in buffer III (5 mM HEPES-KOH pH 7.5, 9 mM MgOAc, 50 mM KCl, 10 mM NH4 Cl, and 1 mM DTT), 2 \(\upmu\)L of each complex was applied to glow-discharged holey carbon grids (QUANTIFOFIL R2/2), then flash-frozen in liquid ethane using a Vitrobot Mark III (FEI). The grids were transferred to a Cs-corrected Titan Krios electron microscope (FEI) operating at 300 kV. Using FEI's EPU software 1.4.0, images were automatically recorded on their Falcon II direct electron detector set to a nominal magnification of 59,000\(\times\) (corresponding to a calibrated pixel size of 1.16 A), and a defocus range of \(-\)1.5 to \(-\)3.0 \(\upmu\)m. The images were acquired in movie mode during exposures of 1 s (corresponding to 7 frames), under low-dose conditions (50 e\({}^{-}\)/A\({}^{2}\)). Semi-automatic particle picking and reference-free 2D classification for discarding defective particles were both done using CryoSPARC 2.9 software [50], with 174,052 particles extracted from 922 micrographs. All subsequent steps used RELION 3.0.6 [51]. The original image stacks were corrected for drift and beam-induced motion, and summed using that software's MotionCor2-like approach. For each micrograph, the contrast transfer function parameters were determined using CTFFIND 4.1.13 [52]. The initial 3D reconstruction was done using the 30S-portion of the crystal structure of the _E. coli_ ribosome (PDB 4V4Q) [53] as the initial reference. This reference model was first low-pass filtered to a resolution of 40 A to prevent bias toward high-frequency components in the map. The particles were then split into eight classes. As previously described [54; 55], these classes represent several stages of 30S biogenesis, with the small subunit (SSU) body almost completely assembled, but the head at different levels of assembly. Unfortunately, the classes suffer from preferential-orientation artefacts. To further study the late stages of the 30S assembly, we combined the three classes that most resemble a mature 30S (subpopulation group IV). The resulting 48,873 particles were submitted to a second round of 3D refinement, followed by CTF refinement and polishing. A final round of 3D auto-refinement and post-processing produced a map with a global resolution of 5.9 A with a Fourier shell correlation of 0.143. The local resolution map was estimated using Resmap 1.1.4 [56]. The consensus map was then used as a basis for multibody refinement [57] into two separate maps of the body and head of the SSU. The corresponding masks were made using a 30 A low-pass filtered version of the consensus map, with soft edges of 12 pixels to define the solvent region boundary and ensure the overlapping of the two bodies. The resulting density maps were then sharpened using Phenix [58] and fitted on the consensus map using UCSF-Chimera [59]. To interpret the map, UCSF-Chimera was used to rigid-body fit the SSU-portion of the crystal structure of the _E. coli_ (PDB 4 YBB) mature 70S subunit [60] into the cryo-EM maps, with each protein and RNA treated separately. The atomic models of the protein and rRNA were then manually adjusted using COOT [61]. The entire model was further improved by real-space refinement against the consensus map using Phenix. The remaining analysis and the illustrations were undertaken using UCSF-Chimera. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ijms24043491/s1](https://www.mdpi.com/article/10.3390/ijms24043491/s1)]([https://www.mdpi.com/article/10.3390/ijms24043491/s1](https://www.mdpi.com/article/10.3390/ijms24043491/s1)). [PERSON] performed cryo-EM analysis and analyzed data; [PERSON] and [PERSON] performed biochemical experiments; [PERSON] and [PERSON] conducted 21S purification experiments; [PERSON] conducted rRNA experiments; and [PERSON] and [PERSON] performed the proteomic analyses. [PERSON] and [PERSON] conceived the project, designed the experiments, and wrote the manuscript.