4. Acquisition

Time requirement: 42 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-gradient 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 in F₁ and F₃, and the frequency dimension for ¹⁵N is F₂. The sequence used here switches the proton offset from H_α/β at the beginning to water after gradient g2, for better performance. Record a 1D proton NMR spectrum and adjust the offset on water. You have to set:

td3: 8k data points in F₃ (¹H)
td2: 48 data points in F₂ (¹⁵N)
td1: 150 data points in F₁ (¹H)
sw3: 11 ppm
sw2: 40 ppm
sw1: 9 ppm
o1: middle of H_α/β region [3.5 ppm] of ¹H spectrum and switch to water resonance
o2: middle of ¹³C NMR spectrum (C_α/β region) [42 ppm and offset switching to the carbonyl region [175 ppm] for the selective pulses
o3: middle of ¹⁵N NMR spectrum (amide region) [117 ppm]
p1, p3, p4, p6, p8: 90° ¹H transmitter pulse [8 μs, 5 dB]
p2, p5, p7, p9: 180° ¹H transmitter pulse [16 μs, 5 dB]
p11, p13, p15: 90° ¹⁵N decoupler pulse [30 μs, 2 dB]
p10, p12, p14, p16: 180° ¹⁵N decoupler pulse [60 μs, 2 dB]
p17, p19, p23: 90° band-selective ¹³C decoupler pulse, offset on C_α/β, q5 Gaussian cascade [400 μs, 1 dB]
p18, p20, p22, p24, p25: 180° band-selective ¹³C decoupler pulse, offset on C_α/β, q3 Gaussian cascade [256 μs, 0.5 dB]; note the o2 offset switch in the pulse program after g3; therefore p24 and p25 need a different offset definition and are centered only on C_α
p21: 90° band-selective ¹³C decoupler pulse, offset on C_α/β, q5 Gaussian cascade, time-reversed shape to p19 [400 μs, 1 dB]
p28: 90° band-selective ¹³C decoupler pulse, offset on CO, q5 Gaussian cascade [400 μs, 1 dB]
p30: 90° band-selective ¹³C decoupler pulse, offset on CO, q5 Gaussian cascade, time-reversed shape to p28 [400 μs, 1 dB]
p26, p27, p29, p31: 180° band-selective ¹³C decoupler pulse, formal offset on CO, q3 Gaussian cascade [256 μs, 0.5 dB]

In the pulse sequence used, after g2 in the proton channel and after g3 in the ¹³C channel the offsets are switched from H_α/β to water, and from C_α/β to CO, respectively. For this you have to provide two frequency lists,