Extended Data Fig. 3: Domain level variability in map quality and molecular flexibility.

(a) Local resolution as calculated by cryoSPARC is colored from high local resolution (blue) to low local resolution (orange) on the sharpened 2.2 Å map at 3σ threshold. (b) The 2.2 Å model is colored by regions of high Q-score (blue) and low Q-score (orange). Several factors may contribute to the variation in local resolution and atomic resolvability, including specimen preservation, radiation damage, inherent flexibility, charge distribution, and spatial geometry. Due to the repeated structural arrangement and relatively uniform solvent accessible of RNA, we hypothesized that inherent flexibility was the major source of variation in local resolution and atomic resolvability across the ribozyme. (c-d) Flexibility of the structure with flexible regions colored orange, as indicated by (c) Root Mean Square Deviation (RMSD) of all heavy atoms between the 2.2 and 2.3 Å models, and (d) Root Mean Square Fluctuation (RMSF) of all heavy atoms estimated from 30 400 ns MD simulations. (e) Plot of per-residue Q-score and RMSF with domain colors matching Fig. 1. (f-g) For each nucleotide, (f) the RMSF of the simulations and (g) the B-factor of the simulations (\(\frac{8{\pi }^{2}}{3}{{\rm{RMSF}}}^{2}\)) of the nucleotide in MD simulation is plotted against the average Q-score of the nucleotide in the 2.2 and 2.3 Å model. Spearman’s rank correlation coefficient is reported, with a strong statistically significant negative correlation, calculated with a two-sided hypothesis test for no ordinal rank between the variables (P = 1.1 × 10⁻¹¹⁶), supporting the hypothesis that inherent flexibility is the primary attribute explaining poor resolvability in peripheral regions of the ribozyme. The 2.2 and 2.3 Å models differed most in these poorly resolved, highly flexible regions; the classification scheme used in the image processing successfully distinguished the differences in the highly flexible regions of the RNA. (h) The Spearman’s rank correlation coefficient was calculated between pairs of per-residue values, including the additional values of average local resolution value at the nucleotide position, RMSD between the 2.2 and 2.3 Å models, and the Q-score of the average structure from simulation in a simulation density map (simQ).