Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach

Abstract

Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]₂; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and ²H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.

Keywords: HIV-1 dimer initiation site; NMR; RNA; cryo-EM; molecular dynamics.

Figure 1. Single particle cryo-EM of HIV-1 [DIS]₂

(A) HIV-1 [DIS]₂ RNA sequence. (B) Representative raw cryo-EM micrograph. (C) Boxed raw particles of [DIS]₂ from cryo-EM micrographs. (D) Reference free 2D class averages. (E) Final 3D structure in two views. (B–E: Initial data set imaged on JEM2200FS with DE12 detector.) (F) Final 3D structure reconstructed in two views from second data set (imaged on JEM3200FSC with K2 detector).

Figure 2

(A) Portions of the 2D NOESY spectrum obtained for G⁸C^6,r-labeled [DIS]₂ showing well-resolved G(i)-H8 to C(i-1) and C-H6 to C-H1′ NOEs (bottom) and weak C-H6 to C-H5 breakthrough NOEs resulting from partial C5-deuteration (~90%) (top). The top panel is displayed at 5-fold increased intensity compared to the bottom panel. (B) Portions of the A^2,8G^H-labeled [DIS]₂ 2D NOESY spectrum highlighting NOEs that help identify the S-turn motif (dark labeling). Labels denote NOEs associated with A- or G-H8 protons unless otherwise noted.

Figure 3. MD simulations and implications for the cryo-EM resolution limit

Maps were reconstructed from simulated random projections of MD simulated models. (A) Reconstruction of noise free simulation from 15 discrete MD timesteps at 300K mimicking different structure conformations. (B) Reconstruction from a nearly static [DIS]₂ structure at close timesteps with dynamic explicit solvent noise. (C) Reconstruction with additional 8σ noise, comparable in overall contrast to cryo-EM images.

Figure 4. Structure of HIV-1 [DIS]₂ derived by hybrid NMR/cryo-EM

(A) View of the 20 refined [DIS]₂ structures relative to the 9 Å cyro-EM map. The location of the bulged G241 base is well defined. (B) Surface representation of HIV-1 [DIS]₂ that highlights the super-helical twist and features of the major and minor grooves (phosphates colored red). (C, D) Expanded views of the A-rich bulge (C) and S-turn (D) showing key NOEs (yellow dashed lines) that helped define these structures.