Figure 4.

X-filtering. ¹³C-filtered NOESY spectra of the ²H/¹³C/¹⁵N-U-labeled sample, demonstrating the spectral simplification and improved identification of NOE contacts achieved via this new labeling method, where specific deuteration is combined with specific ¹³C-labeling of crucial residue types. Because in the U residues only H2′ are left on the ribose, otherwise inaccessible NOEs can now specifically be assigned. Subspectrum (A) shows the NOE contacts between ¹³C-bound (f₁) and ¹³C-bound (f₂) protons, e.g. between uridine H2′ and H6 protons (either intra-residue or inter-residue). The intra-residue H6/8-H2′ of U12, U14 and U23 can be seen, because they are partially or completely S-puckered, in contrast to the other U residues (Table S2). In addition, an intense sequential contact is seen between H6 of U19 and H2′ of U18. Subspectrum (B) shows cross peaks between ¹²C-bound protons (f₁) and ¹³C-bound protons (f₂). In the H6/8 (f₁)-H2′ (f₂) region (below the diagonal), two intense cross peaks are seen. They are the sequential H6/8_i-H2′_i–1 of C6-U5 and G20-U19. The two weaker cross peaks are the sequential H6/8_i-H2′_i–1 of G13-U12 and G24-U23. In the H6/8 (f₂)-H2′ (f₁) region (above the diagonal), sequential cross peaks from H2′_i–1 (f₁) to H6/8_i (f₂) can be seen, i.e. from C4-U5, G13-U14, C17-U18 and, weakly, G22-U23. Subspectrum (C) shows cross peaks between ¹³C-bound protons (f₁) and ¹²C-bound protons (f₂). Subspectra C and B are symmetry-related, i.e. each cross peak in C appears in B on the opposite side of the diagonal. Subspectrum (D) shows the ¹²C-bound-to-¹²C-bound contacts, i.e. the NOE contacts between the non-U residues. Note the crowded H2′-H5′/5″ region.