Figure 3. The Putative Structural Model of the RRE, Fitness to the Experimental SAXS Data, Pairwise RMSD of the Top 20 Ensemble Structures, and Histograms of Characteristic Distance Distributions of the RRE RNA.

(A) Superimposition of the putative RRE structural model in red ribbon with the SAXS envelope in cyan mesh. The four-way junction, three-way junction, and the two known primary Rev-binding sites are marked. The distance between the two binding sites is about 55Å. The points A and B on the top of the envelope are also labeled. Domain II structure was generated based on its homology to an adenine riboswitch (Figure S4).

(B) Left: The back-calculated scattering curves with Ne = 3 (blue) superimposed on the experimental SAXS-WAXS (black with error bars) curves. The inset shows the χ² between experimental and back-calculated SAXS-WAXS curves versus the ensemble size, Ne, used in the calculation; top right: an expanded high-q region of the superimposed scattering curves on the left; bottom right: residual differences in scattering curves between the top 20 structures in the ensemble and the average experimental data.

(C) Pairwise RMSD of the top 20 ensembles with Ne = 3. The RMSD of residue j between structures a and b is defined as:

$$\mathit{RMSD}(j)=\mathrm{RMSD}(a^j,b^j)=\sqrt{\frac{1}{N}\sum _{i=1}^N{(a_{ix}^j-b_{ix}^j)}^2+{(a_{iy}^j-b_{iy}^j)}^2+{(a_{iz}^j-b_{iz}^j)}^2}$$

where N is the number of heavy atoms in residue j. The average RMSD (blue dots connected with blue solid line) and SD (red error bars) of residues are calculated for the top 20 ensembles. Residues in duplexes are labeled with black thick lines at the bottom of the plot. The pattern of the pairwise RMSD almost parallels that of reactivity by chemical probing (Legiewicz et al., 2008).

(D and E) Histograms of (D) the distance between the duet of the binding sites IIB and IA, D_duet and (E) between points A and B, D_AB, at the top of the “A” of the ensembles (see Figure 3A). The distances between the centers of mass of phosphate atoms of residues (47G, 70G, 46G, 72A)_IIB and (24G, 206A, 25G, 205G)_IA are taken as the approximate distance between the IIB and IA duet of binding sites, while the locations of A and B are defined as the centers of the phosphates of residues 16G and 225U. The red lines are the best fits to the histograms assuming a Gaussian distribution. See also Figures S3 and S4 and Movies S3 and S4.