Fig. 4.

Panel a): pulse sequence of ¹³C-edited 3D SFNOESY-HMQC for frequency labeling H_M(F₁)-C_M(F₂)H_M(F₃) or H_N,Aro-C_MH_M (with addition of two ¹⁵N 180° hard pulses at the t₁ and δ midpoint). The smaller and larger ¹H shaped pulses are 1.69 ms long 90° PC9_4_90 and 1.15 ms long 180° REBURP, respectively. The flip angle (α) of the shaped pulse after τ_mix should be optimized as discussed in maintext. The first and second vertical arrows indicate the ¹H carrier frequency setting to 0.9 ppm for C_M(F₂)H_M(F₃) (8.5 ppm for H_N,Aro-C_MH_M) and 4.7 ppm, respectively. The offsets of ¹H shaped pulses before and after τ_mix are 0 Hz (at 4.7 ppm) and −3,230 Hz (at 0.9 ppm), respectively. Please note that two refocusing ¹⁵N 180° hard pulses at the midpoint of t₁ and δ should be added for H_N,Aro-C_MH_M. The spectral centers of ¹H(F₁), ¹³C(F₂), ¹H(F₃) dimensions are at 1.0, (8.5 for H_N,Aro-C_MH_M), 16.5, and 4.7 ppm, respectively. The delays are: d1 = 200 ms, δ = t₁(0) + 2×pwN (initial t₁ value and ¹⁵N 180° pulse width), Δ = 4.0 ms, τ_mix = 300 ms. The phase cycling are: φ1 = (x, −x), φ2 = (x, −x, −x, x), φ3 = 4(x), 4(y), φ_rec = (x, −x, −x, x, −x, x,×−x). Bruker decoupling scheme bi_garp_2pl is used. The quadrature detections in t₁ and t₂ dimensions are acquired via States-TPPI of φ1 and φ2, respectively. The durations and strengths of the gradients are G1 = (1 ms, 15 G/cm), G2 = (1 ms, 5 G/cm). Comparisons of ¹³C-resolved 3D NOESY-HMQC with ¹³C-resolved SFNOESY-HMQC counterpart are shown in b) and c) left and right strips, respectively. Experiments were run on ¹⁵N-methyl-aromatic labeled FliT-FliJ sample and processed in an identical manner. In the reference spectrum the full 11ppm indirect ¹H dimension is sampled (H_All) while in the SOFAST sequence the acquisition is split into two 3Ds, one centered on the indirect-detected amide/aromatic (H_NH_Aro) (b), and one on the methyl (H_M) ¹H chemical shift (c). Note that the same 3D-NOESY-HMQC strip marked as ‘reference’ is split into two halves for side-by-side comparison. In (d) the number of NOE per residue (and totals) resulting from CYANA automated NOESY assignment protocol for the FliT-FliJ protein are shown for the ‘reference’ (upper) and SOFAST datasets (lower).