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Experiment Details: 3D triple cross polarization 13C15N13C correlation experiment
Dimensions = 3
Isotope labeling required = 13C and 15N
Magnetization transfer = magnetization originates on 1H and is transferred to 13Cα via cross polarization and then selectively to 15N nuclei through specific CP. An additional CP step is added to transfer magnetization to 13CO. Finally a DARR step is used to transfer magnetization to neighboring 13C nuclei. Chemical shift evolves on 13Cα, 15N and 13CO and detected on 13C.
Image courtesy: www.protein-nmr.org.uk/
Pulse program: hCaNCOCX3D.tcp
Parameters:
p1 : C 90 at pl1
p2 : C 180 at pl1
p3 : H 90 at pl2
p15 : HC CP at sp41 (f1,C) and sp40 (f2,H)
p16 : NCa CP at pl5 (f3,N), sp50 (f1,C), pl33 (f2,H)
p17 : NCO CP at pl6 (f3,N), sp51 (f1,C), pl34 (f2,H)
p21 : N 90 at pl21
p22 : N 180 at pl21
pl1 : C pulse power
pl2 : H pulse power
pl5 : N NCa CP power
pl6 : N NCO CP power
pl12 : H dec power
pl14 : H DARR power (= 1*cnst31 (MAS freq); for PDSD = 0 W)
pl21 : N pulse power
pl33 : H dec power during NCa CP
pl34 : H dec power during NCO CP
sp40 : H CP power
sp41 : C CP power
sp50 : C NCa CP power
sp51 : C NCO CP power
d0 : incremented delay (t1)
d1 : recycle delay; 1 to 5 times T1
d8 : PDSD/DARR mixing time
d10 : incremented delay (t2)
d29 : extra time for constant duty cycle (t2)
d30 : extra time for constant duty cycle (t1)
pcpd2 : H dec pulse cpdprg2
cpdprg2 : Decoupling file cw, tppm (at pl12)
spnam40 : H ramp use e.g. ramp.10070 for variable amplitude CP
spnam41 : C ramp use e.g. square.100 for square pulse CP
spnam50 : CA ramp use e.g. tcn for amplitude modulated tangential pulse CP
spnam51 : CO ramp use e.g. tcn for amplitude modulated tangential pulse CP
cnst10 : Frequency reset to Carrier (o1, usually ~100 ppm)
cnst21 : Frequency offset for CO (~175 ppm)
cnst22 : Frequency offset for CA (~55 ppm)
cnst29 : expected td2 for use in constant duty cycle (-DCDC)
cnst30 : expected td1 for use in constant duty cycle (-DCDC)
cnst31 : MAS rotation rate in Hz
inf1: 1/SW(Ca) = 2 * DW(Ca)
inf2: 1/SW(N) = 2 * DW(N)
in0 : = inf1
in10: = inf2
in29: = inf2
in30: = inf1
l0 : loopcounter for F1
l1 : loopcounter for mixing time
l10: loopcounter for F2
ZGOPTNS : -DCDC : for constant duty cycle (parmode must equal DIM)
-DnoMix : to eliminate COCx mixing or blank
FnMODE : TPPI, States or States-TPPI
ns: MIN. 4 (full:16)
Comment on key optimization:
cnst22 = '55'; cnst21 = '175; p2 = 2*p1; p22 = 2*p21;
CNST31 = wr;PLW14 = PLW12 found in DARR optimization
parmode = 3D; o1p = '100'; o2p = '3'; o3p = '115'; ZGOPTNS = '-DCDC' for constant duty cycle
Example spectrum of GB1: The spectrum is highly useful in overcoming incomplete assignments problems, due to spectral overlap. The experiment is used for Cαi-Ni-COi-1-Cαi-1 links. A major drawback of the experiment is the low signal to noise which will affect detection of signals for a sample which already suffers from a low signal to noise ratio.
1-3 plane
1-2 plane
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