Fig. 3: Determining the conformational ensemble of TAR^ES2.

a Secondary structure of TAR^ES2 and an elongated construct (E-TAR^ES2) obtained by inserting residues (in gray) at the indicated position in the lower helix. b Comparison of the RMSD between measured and predicted RDCs (top, TAR^ES2 + E-TAR^ES2, dashed line corresponds to uncertainty) and chemical shifts (bottom, measured on wtTAR ES2) for the FARFAR-Random (deep blue), FARFAR-Lowest (light blue), FARFAR-NMR (purple), and FARFAR-MD-NMR (magenta) ensembles. Bulge refers to chemical shifts measured at residue U23. c, d Comparison between measured and ensemble predicted (c) representative chemical shifts measured on wtTAR ES2 (The outlier A-C1’ chemical shifts are highlighted in open green symbols) and (d) RDCs (TAR^ES2 + E-TAR^ES2). Also shown is the number of conformers (N) in the ensemble as well as the R² and RMSD between measured and predicted values. RDCs and chemical shifts are color-coded according to the structural elements in (a). e Structural overlay of the FARFAR-Random, FARFAR-NMR, and FARFAR-MD-NMR ensembles (N = 10) along with a zoomed-in view of the tandem AG mismatches and bulge motif. Partially melted, high-χ angle (−130 ± 20°), and regular χ-angle (<−150°) A-G mismatches are colored purple, yellow, and green, respectively. f Overlay of conformers showing inter-helical stacking accompanied by the flipping out of bulge residue U23. g Secondary structure of the ten conformers in the FARFAR-MD-NMR ensemble. Residues with C2’-endo sugar pucker and non-gauche + γ (falling outside 20-100°) torsion angle are highlighted using blue filled and orange open circles, respectively. Watson–Crick bp and Wobble bp are denoted using a solid line, whereas other bp geometries are indicated as a dashed line. h Structural overlay of mismatches in the FARFAR-MD-NMR ensemble with bases used as a reference for superposition colored pink.