4. Acquisition

Time requirement: 20 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 on the second. Note that the software uses different numbering; here protons are not in the first dimension, but in F3; the frequency dimension for ¹³C_α/β is F1 and that for ¹⁵N is F2. Record a 1D proton NMR spectrum with water suppression and adjust the offset in the middle of the amide region. You have to set:

td3: 1024 data points in F3 (¹H)
td2: 32 data points in F2 (¹⁵N)
td1: 32 data points in F1 (¹³C_α/β)
sw3: 4.2 ppm
sw2: 40 ppm
sw1: 70 ppm
o1: middle of ¹H spectrum (amide region) [7.8 ppm]; for improvement of water suppression it is also possible to set o1 to the water frequency and to use the full proton spectral width.
o2: middle of ¹³C NMR spectrum (C_α/β region) [42 ppm]
o3: middle of ¹⁵N NMR spectrum (amide region) [117 ppm]
p1, p3, p5, p7, p9: 90° ¹H transmitter pulse [8 μs, 5 dB]
p2, p4, p6, p8, p10: 180° ¹H transmitter pulse [16 μs, 5 dB]
p13, p15, p17: 90° ¹⁵N decoupler pulse [30 μs, 2 dB]
p11, p12, p14, p16, p18: 180° ¹⁵N decoupler pulse [60 μs, 2 dB]
p19, p21, p25: 90° band-selective ¹³C decoupler pulse, offset on C_α/β, q5 Gaussian cascade [400 μs, 1 dB]
p20, p22, p24, p26, p27: 180° band-selective ¹³C decoupler pulse, offset on C_α/β, q3 Gaussian cascade [256 μs, 1 dB]
p23: 90° band-selective ¹³C decoupler pulse, offset on C_α/β, q5 Gaussian cascade, time-reversed shape to p20 [400 μs, 1 dB]
p32, p34: 90° band-selective ¹³C decoupler pulse, offset on CO, q5 Gaussian cascade [400 μs, 1 dB]
p28, p29, p30, p31, p33, p35: 180° band-selective ¹³C decoupler pulse, offset on CO, q3 Gaussian cascade [256 μs, 1 dB]
In the pulse sequence used, after p25 in the ¹³C channel the offset is switched from C_α/β to CO. For this you have to provide a frequency list.
d1: 2 s
d2: 1/[4J(C,H)] = 1.8 ms, calculated from ¹J(C,H) = 140 Hz
d3: 1/[3J(C,H)] = 2.2 ms − p23/2
d4: 3.6 ms − d3 − p23, constant time period for C_α/β
d5: 3.6 ms, decremented during constant-time period for C_α/β