Fig. 5.

Pulse sequence for the 3D jnJ-scaled TROSY-HNCO experiment. Scheme n is inserted at the position labeled n = −1 for recording the upfield line and at the position labeled n = 1 for recording the downfield line. All narrow (wide) pulses are applied with a flip angle of 90° (180°) with a phase of × unless otherwise indicated. ¹H and ¹³C carriers are centered at the water resonance and 175 ppm, respectively, and off-resonance ¹³C^α pulses were centered at 55 ppm. The ¹⁵N carrier was adjusted for each scaling scheme and centered at 117.5, 118.5, 116.5 and 119.5 ppm for values of nj of −1, 1, −2 and 2 respectively. ¹H and ¹⁵N pulses are applied with a pulse width of 9.3 and 38 ls, respectively, except for the rectangular water selective pulses which have a pulse width of 1.16 ms. Rectangular ¹³C 90° pulses are applied with a pulse width of 14 μs, and shaped ¹³C inversion pulses are applied with a g3 profile (225 μs at 800 MHz) (Emsley and Bodenhausen 1990). The delays used are: τ_α = 2.5 ms, T_N = 14 ms, τ_β = 29.1 μs, δ = 3 μs. The parameter κ determines the extent of J scaling with apparent coupling J_app = κ × ¹J_NH. The parameters α and β are set such that α + β = κ with α = κ when κ ≤ 1 and α = 1 when κ > 1. The following eight step phase cycle is employed: ϕ₁ = y, −y, −x, x; ϕ₂ = x; ϕ₃ = 4(x), 4(−x); ϕ₄ = −y; ϕ₅ = y; ϕ₆ = y; ϕ_rec = x, −x, y, -y, −x, x, −y, y. Quadrature is achieved in F1 by the method of States-TPPI on ϕ₂ and in F2 by the echo-antiecho (enhanced sensitivity) (Rance et al. 1999) method whereby ϕ₄, ϕ₅, ϕ₆ and the latter two phases of ϕ₁ are incremented by 180° for each complex t₂ point. The initial duration of t₂ is set at 1/2SW, which requires a 180° first order phase correction during processing. The gradients employed are: g1 (0.5 ms, 2.16 G/cm), g2 (0.2 ms, 21.56 G/cm), g3 (0.2 ms, 15.1 G/cm), and g4 (0.3 ms, 32.35 G/cm)