3D HN(CA)CO

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• Insert the sample.
• Choose the solvent deuterium signal with the lock command.
• Tune and match the probehead.
• Optimize the shim procedure (read a shim file with the rsh command if required).

Create a new dataset with edc or, alternatively, increment the experiment number with the iexpno command.

Read the standard parameter set necessary to record a gradient-based sensitivity-enhanced 3D HN(CA)CO experiment by typing rpar HNCACOGP3D all (the hncacogp3d pulse program can be displayed with the edcpul command). Otherwise, the pulse scheme can be visualized using showpp.

Channel	f1	f2	f3
Nucleus	1H	13C	15N
Offset	o1p	o2p	o3p
Carrier	4.75 ppm	172.5 ppm	115.5 ppm

 

The following hard and shaped pulses applied on the three channels must be previously calibrated:

• Selective 90 ¹³C pulses applied via the decoupler f2 channel:
  • On-resonance of the CA resonances (p13 (about 409u) at sp2, using spnam2=G4.256, spoff2=0)
  • On-resonance of the CO resonances (p13 (about 409u) at sp8, using spnam8=G4tr.256, spoff8=0).
• Selective 180º ¹³C pulse applied via the decoupler f2 channel:
  • On-resonance of the CA resonances (p14 (about 256u) at sp3, using spnam3=G3.256, spoff3=0). A second, higher-selective on-resonance CA pulse is also involved (p24 at sp9, using spnam9=G3.256, spoff9=0).
  • Off-resonance of the CO resonances (p14 (about 256u) at sp5, using spnam5=G3.256, spoff5=+121ppm)
  • Off-resonance of the CA resonances (p14 (about 256u) at sp7, using spnam7=G3.256, spoff7=-121ppm)
• Hard 90º and 180º ¹⁵N pulses via the decoupler f3 channel (p21 and p22 at pl3).
• 90º ¹⁵N pulse for GARP decoupling during acquisition via the decoupler f3 channel (pcpd3 at pl16 using cpdprg3=garp).

Otherwhise, all required acquisition parameters can be displayed with the ased command. By default:
 

 

Dimension	F3	F2	F1
Nucleus	1H	15N	13C
TD	2048	40	128
Spectral width	NH  (14.08 ppm)	N  (32 ppm)	13CO (22 ppm)

 

Interpulse delays:

d21 is optimized to 1/(2*J_N-H) (5.5 ms)           
d22 is optimized to 1/(4*J_CA-CO) (4 ms)
d23 is optimized to 1/(4*J_N-CA)) (12 ms)
d26 is optimized to 1/(4*J_N-H) (2.3 ms)

All other delays are automatically calculated from these values. Gradient parameters are already defined and the user must be set the number of scans and dummy scans (by default, ns=4 and d2=8) as a function of sample concentration. This experiment contains 5 gradients and the standard parameters are already defined with p16 of 1ms, d16=100u, gpnam1-3=sine.100 and the ratio is 30:80:8.1.

A frecuency list FQ2LIST must be defined (list has to be stored in the directory) in which the CA frequency (52 ppm) is set for first part and the CO frequency (173 ppm) is set for second part.

Start acquisition by rga and zg (the expected experimental time can be observed with the expt command).

Process the recorded data with tf3, tf2 and tf1. The standard processing parameters are set to:
 

Dimension	F3	F2	F1
SI	2048	256	512
MC2	-	echo/antiecho	States-TPPI
WDW	QSINE	QSINE	QSINE
Offset	2	2	2
ME_mod	no	LPfc	LPfc
NCOEF	0	32	32

 

 Use XWINPLOT.

It is advisable to store all acquisition and processing parameters (for instance, with the wpar hncacogs3d all command) to be used later.

List of available 3D HN(CA)CO versions.

Any version of the 3D HNCA experiment can be automatically recorded as a 2D experiment. The only thing to do is to set the number of experiments (TD) in one of the two indirect dimensions to 1 (eda window). After start acquisition with zg, data can be processed from the 3D menu display using xfb (the number of procno must be typed). In this way, the 2D H(NCA)CO and the 2D HN(CACO) experiments can be recorded under the same experimental conditions. In fact, these are the two first 2D planes of the 3D data.

Related 3D Experiments.