To further aid in the assignment of the observed E states, we performed MD simulations of the DNA construct used in the single-molecule experiments. DNA constructs containing hybrid 1, hybrid 2, 2-tetrad basket and chair (wild-type and mutant) G4s were simulated (Figure 2E and ‘Materials and Methods’ section for details). We find that the G4 structures are predominantly positioned on top of the duplex, i.e. with the G-tetrad plane perpendicular to the long axis of the duplex, for the DNA constructs containing the hybrid 1, hybrid 2 and chair (wild-type and mutant) conformations. However, the 2-tetrad basket conformation is predominantly tilted relative to the duplex, resulting in positioning of the G-tetrad plane almost parallel to the long axis of the duplex (Supplementary Figure S7). This feature is likely to originate due to the structural specificity of the 2-tetrad basket conformation that contains a long diagonal loop that may create some steric restrictions for the G4-duplex relative positioning. To assess the transfer efficiency for each simulated G4 construct we used the AV approach (39) (Figure 2E, Supplementary Figure S5 and ‘Materials and Methods’ section for details). The transfer efficiencies derived from the simulations can be directly compared to those obtained from single-molecule FRET experiments (Figure 2F). The experimental and simulated transfer efficiencies for the chair, hybrid 1 and hybrid 2 conformations match within uncertainty. We even observe a small difference in the simulated transfer efficiencies for the mutant (E = 0.84) and wild-type (E = 0.89) chair conformations (Supplementary Figure S5), that is also detected in the single-molecule experiments (Figure 2). This reflects the small structural differences of the two constructs and demonstrates the robustness of both approaches of assessing the transfer efficiencies. Due to the specific positioning of the 2-tetrad basket conformation relative to the duplex, the separation between the two fluorophores is larger than for other G4 conformations. It appears in a range very close to the Förster radius (R0 = 5.6 Å) (Supplementary Figure S5), where the energy transfer efficiency is mostly sensitive to distance changes. This observation can potentially explain the small discrepancy between the experimental and simulated transfer efficiencies for this particular conformation. Overall, we find a remarkable agreement between the two sets of transfer efficiencies obtained from single-molecule FRET experiments and simulations, which further supports our structural assignment.