Visualizing RNA Structures by SAXS-Driven MD Simulations

Abstract

The biological role of biomolecules is intimately linked to their structural dynamics. Experimental or computational techniques alone are often insufficient to determine accurate structural ensembles in atomic detail. We use all-atom molecular dynamics (MD) simulations and couple it to small-angle X-ray scattering (SAXS) experiments to resolve the structural dynamics of RNA molecules. To accomplish this task, we utilize a set of re-weighting and biasing techniques tailored for RNA molecules. To showcase our approach, we study two RNA molecules: a riboswitch that shows structural variations upon ligand binding, and a two-way junction RNA that displays structural heterogeneity and sensitivity to salt conditions. Integration of MD simulations and experiments allows the accurate construction of conformational ensembles of RNA molecules. We observe a dynamic change of the SAM-I riboswitch conformations depending on its binding partners. The binding of SAM and Mg²⁺ cations stabilizes the compact state. The absence of Mg²⁺ or SAM leads to the loss of tertiary contacts, resulting in a dramatic expansion of the riboswitch conformations. The sensitivity of RNA structures to the ionic strength demonstrates itself in the helix junction helix (HJH). The HJH shows non-monotonic compaction as the ionic strength increases. The physics-based picture derived from the experimentally guided MD simulations allows biophysical characterization of RNA molecules. All in all, SAXS-guided MD simulations offer great prospects for studying RNA structural dynamics.

Keywords: MD simulation; RNA; SAXS (small-angle X-ray scattering); experimentally guided simulations; maximum entropy; structural modelling.

FIGURE 1

Flowchart of generating experimentally consistent RNA structures using small-angle x-ray scattering (SAXS) data. (A) The atomic model of target biomolecule is built or taken PDB/NDB. The step of the SAXS assessment after molecular modeling sorts the structure pool and picks out the best-fit conformations determined by the agreement (the threshold χ ² ≤ 20). Different SAXS-driven strategies are used depending on RNA: a single-replica protocol for RNAs that are relatively well-structured, parallel-replica protocol for flexible RNA constructs. (B) Illustration of the RNA + ion + water and water + ion systems used to compute SAXS. The molecular envelope is constructed from a three-dimensional probability density isosurface 10 Å from the molecular surface to encompass the solvent/ion shell. The scattering intensity of the water + ion system was computed by applying the same envelope. We subtract the scattering from the RNA-ion system from solvent as described in Section 2.

FIGURE 2

Structure and scattering profile of SAM-I riboswitch aptamer. (A) Secondary and (B) tertiary structure of the ligand-bound SAM-I aptamer. Key tertiary interactions are labeled as follows: P, paired; J, joining; KT, kink-turn; and PK, pseudoknot. The helices P1–P4 are colored in the tertiary structure according to the secondary structure representation. The ligand SAM is colored in red, and the SAM-binding residues are shaded in light red. (C) Kratky representation of the experimental scattering data that were used as a target structure for the SAXS-driven MD of the riboswitch under different configurations [s1: SAM(+),Mg²⁺(+); s2: SAM(−), Mg²⁺(+); s3: SAM(−), Mg²⁺(−)].

FIGURE 3

SAM-I riboswitch aptamer in the presence of SAM and Mg²⁺. (A) Time evolution of χ ² variable during SAXS-driven MD simulation. (B) Converged structure after SAXS-driven MD simulation with experimental data for the Mg²⁺- and SAM-bound aptamer state. Intensity in the (C) normal and (D) Kratky representation. The experimental SAXS data (gray), computed SAXS data of the crystal structure (magenta), and computed SAXS data of the SAXS-driven MD final structure (blue). (E) Comparison of the SAXS-driven MD structure (blue) with the crystal structure (magenta).

FIGURE 4

Apo-state of the SAM-I riboswitch aptamer in the presence of Mg²⁺. (A) Time evolution of χ ² variable during simulation. (B) The final structure from SAXS-driven MD. (C) Kratky plot representation of the molecule, experimental SAXS data (gray), computed SAXS data of the crystal structure (magenta), and computed SAXS data of the SAXS-driven MD structure (blue). (D) Comparison of the SAXS-driven MD structure (blue) and the Mg²⁺-, and SAM-bound crystal structure (magenta).

FIGURE 5

Contact formation frequency between the residues upon the removal of the ligand molecule SAM. Contact map of (A) the ligand-bound (s1) and (B) ligand-free (s2) SAM-I riboswitch. The map is based on a 6-Å cutoff for the minimum distance between the residues. Interhelical residues that get closer upon removing the ligand are highlighted with green and cyan/blue bars. The junctions J1/2, J3/4, and J4/1 are colored in orange, magenta, and blue-yellow, respectively. The same color scheme is applied in the inserted structural representation to illustrate the location of these contact changes.

FIGURE 6

Comparison of the residue level local Mg²⁺ concentration at the ligand-bound (s1) and apo-state (s2) of the SAM-I riboswitch. The regions of the riboswitch are highlighted in color, P1 (green), P2 (blue), P3 (red), and P4 (orange). The inserted structural representation of s1 highlights the location of the two identified Mg²⁺ binding sites within the helix P2.

FIGURE 7

Apo-state of the SAM-I riboswitch aptamer in the absence of Mg²⁺. Ensembles of conformations obtained by SAXS-driven MD simulations. The final structures (top), time evolution of χ ² (middle), and resultant Kratky plots (bottom) are given for four independent simulations. The intensity of the structure before (magenta) and after (blue) the SAXS-driven MD in comparison to the experimental data (gray).

FIGURE 8

Comparison of the structural dynamics of SAM-I riboswitch at each functional states. (A) RMS fluctuations along the backbone of the RNA in the presence of SAM and Mg²⁺ (s1, black), in Mg²⁺ and without the ligand (s2, blue) and in the absence of divalent and SAM (s3, purple). The regions of the riboswitch, namely, P1, P2, P3, and P4, are highlighted in green, blue, red, and orange, respectively. (B) Structural dynamics is compared by showing the most populated clusters for each condition obtained by SAXS-MD.

FIGURE 9

Structure and sequence of RNA HJH. (A) The HJH molecule consists of three components: 12-bp duplexes that are connected by a rU₅ junction (green). (B) The positioning between the bottom duplex (cyan) and the upper duplex (firebrick) is used to define the two axial vectors measuring twisting and bending. (C) SAXS data from experiments in low (50 mM), middle (100 mM), and high (500 mM) [KCl] shown as Kratky plots.

FIGURE 10

SAXS-driven MD simulation results for the HJH RNA at three concentrations. (A–C) Time evolution of χ ² for (A) 50 mM, (B) 100 mM, or (C) 500 mM KCl. The asymptotic amplitude of χ ² indicates the convergence. (D–F) Comparison of the measured SAXS data and the computed SAXS averaged over trajectory after the simulations converged.

FIGURE 11

Spherical heat maps monitoring the occupancy of conformations visited by the HJH RNA. The conformations are monitored by two angles (bending and twisting) in different salt conditions (A) 50 mM, (B) 100 mM, and (C) 500 mM KCl. The representative structures (D–F) for each salt condition. Each frame represents a cluster center; the most populated cluster is colored in blue.

FIGURE 12

Comparison of radius of gyration (Rg) and efficiency of Förster resonance energy transfer (E _FRET ) values from experiment and modeling. (A) Rg from experiment and SAXS-driven MD and (B) FRET efficiencies from simulation and experiment. Fluorescent label sites are briefly illustrated by orange stars. R₀ = 60 Å was applied for computing the theoretical E _FRET from MD simulations. The raw data and EOM analysis can be found in the works of Sutton and Pollack (2015) and Chen et al. (2019).