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 (DIS2; 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. Display omitted •Subnanometer resolution cryo-EM structure of 30 kDa HIV-1 RNA dimerization signal•The major groove of the RNA duplex was unambiguously resolved in the cryo-EM map•Atomic model of HIV-1 RNA duplex was obtained by integrating NMR, cryo-EM, and MD•Superhelical twist and flipped-out base were observed in this structure Zhang, Keane et al. present the structure of the 30 kDa HIV-1 RNA dimerization signal using a hybrid approach combining global structural information from cryo-EM at subnanometer resolution with atomic resolution local structural information from NMR. Using MD simulations they found that cryo-EM resolution may be limited by internal structural flexibility.