4. Acquisition

Time requirement: 3 h

Sample: 10 mg fully ¹³C- and ¹⁵N-labeled human ubiquitin in 600 μl 90% H₂O/10% D₂O, 50 mM phosphate (potassium salt), pH 5.8, 0.5 mM DSS.

Set and control the temperature to 300 K. A triple-resonance inverse probe-head with z-gradients must be tuned to the sample on all three channels. ¹⁵N is assigned to the third hardware channel and ¹³C to the second. Note that the software uses different numbering; here protons are not in the first dimension, but in F2, and the frequency domain for ¹⁵N is F1. Record a 1D proton NMR spectrum and adjust the offset on resonance of the water signal. You have to set:

td2: 1024 data points in F2 (¹H)
td1: 128 data points in F1 (¹⁵N)
sw2: 12 ppm
sw1: 40 ppm
o1: middle of ¹H spectrum (on resonance of water signal)
o2: middle of ¹³C NMR spectrum (C_α/β region) [43 ppm]
o3: middle of ¹⁵N NMR spectrum (amide region) [117 ppm]
p1, p3, p4, p7: 90° ¹H transmitter pulse [8 μs, 5 dB]
p2: 180° ¹H transmitter pulse [16 μs, 5 dB]
p8, p13: 0.231·p1 (Watergate pulse)
p9, p12: 0.692·p1 (Watergate pulse)
p10, p11: 1.4621·p1 (Watergate pulse)
p5: 90° ¹H transmitter pulse at power level of spin-lock [70 μs, 23 dB]
p6: 90° selective rectangular ¹H transmitter pulse on water [1 ms, 46 dB]
p15, p17: 90° ¹⁵N decoupler pulse [30 μs, 2 dB]
p14, p16, p18: 180° ¹⁵N decoupler pulse [60 μs, 2 dB]
p19, p21, p23: 90° band-selective rectangular ¹³C decoupler pulse, offset on C_α/β [49 μs, 8 dB]
p20, p22, p24: 180° band-selective rectangular ¹³C decoupler pulse, offset on C_α/β [44 μs, 1 dB]
p25: 180° band-selective ¹³C decoupler pulse, offset on CO, g3 Gaussian cascade [256 μs, −1 dB]
d1: 1.7 s
d2: 1/[2J(C,H)] effective gradient duration = 3.5 ms
d3: 1/[4J(C_α,N)] ≈ 11 ms
d4: 1/[4J(C_α,N)] ≈ 11 ms, decremented during constant time period for ¹⁵N
d5: 1/[4J(C_α,N)] ≈ 11 ms
d6: Watergate delay 210 μs
d7: 1/[4J(N,H)] ≈ 2.25 ms optimized for relaxation
g1–g6: sinusoidal-shaped field gradients, ca. 0.1 T/m strength, with gradient loop counters, ring-down delays (50 μs), lock blanking and gradient coil blanking switches according to actual instrumentation used. Gradient duration and strength according to the table: