3D HNCA

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• Insert the sample (ij).
• Choose the solvent deuterium signal with the lock command
• Tune and match the probehead (wobb or atma if required).
• Optimize the shim procedure (read an optimized shim file with the rsh command or performs a gradshim if required).

Create a new dataset (new) and read the standard BRUKER parameter set (rpar) to record a gradient-based sensitivity-enhanced 3D HNCA experiment by typing rpar HNCAGP3D all (the hncagp3d pulse program can be visualized in the PulsProg section or with the edcpul command).

Channel	f1	f2	f3
Nucleus	1H	13C	15N
Offset	o1p	o2p	o3p
Carrier	4.75 ppm	51.67 ppm	115.5 ppm

Update the corresponding pulses and power levels in the acquisition parameters according to the selected solvent/probehead parameters by executing the getprosol command (pulses and power levels must be correctly set by the edprosol command). The following hard and shaped pulses applied on the three channels must be previously calibrated:

Hard 90 and 180 1H pulses (p1 and p2 at high power level pl1) via the observe f1 channel. Selective 90 1H pulse on the water resonance (p11 (about 1.5-2ms) at sp1 using spnam1=Sinc1.1000 and spoff1=0) via the observe f1 channel. Low-power 90 1H pulse for broadband DIPSI-2 decoupling (p26=pcpd1 at pl19 using cpdprg1=dipsi2) via the observe f1 channel. Selective 90 13C pulses applied on the CA resonances via the decoupler f2 channel (p13 (about 409u) at sp2, using spnam2=G4.256, spoff2=0). This same pulse is also applied to perform a time reversed 90 pulse on the CA resonances (p13 (about 409u) at sp8, using spnam8=G4tr.256, spoff8=0). Selective 180 13C pulses applied on the CA resonances via the decoupler f2 channel (p14 (about 256u) at sp3, using spnam3=G3.256, spoff3=0). Selective 180 13C pulse applied off resonance on the CO resonances via the decoupler f2 channel (p14 (about 256u) at sp5, using spnam5=G3.256, spoff5=120ppm). Hard 90 and 180 15N pulses via the decoupler f3 channel (p21 and p22 at pl3). 9015N 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 ppm)	N  (32 ppm)	13CA (32 ppm)

 

The following interpulse delays are defined by default:

            d21 is optimized to 1/(2*J_N-H) (5.5 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. The user must be set the Number of scans (ns) and Dummy scans (ds) as a function of sample concentration.

This experiment contains 3 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

Start acquisition by rga and zg (the expected experimental time is displayed with the expt command). On the other hand, the first 2D planes can be recorded by setting TD=1 in each indirect dimension

The recorded 3D data is Fourier transformed with tf3, tf2, and tf1. Modify processing parameters is required from the ProcPars section. 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 the TOPSPIN plot editor (xwinplot)

It can be advisable to store all acquisition and processing parameters (with the command wpar) to be used later.

This experiment can also be recorded in full-automation mode usin NMR Biotools

List of available 3D HNCA 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(N)CA and the 2D HN(CA) 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.