• 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 HNCACB experiment by typing rpar HNCACBGP3D all (the hncacbgp3d pulse program can be displayed with the edcpul command).

Channel	f1	f2	f3
Nucleus	1H	13C	15N
Offset	o1p	o2p	o3p
Carrier	4.75 ppm	37.36 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 90º and 180º ¹H pulses via the observe f1 channel (p1 and p2 at pl1).
• 90º degree ¹H pulse for broadband DIPSI-2 decoupling via the observe f1 channel (pcpd1 at pl19 using cpdprg1=dipsi2).
• Selective 90º ¹H pulse on the water resonance via the observe f1 channel (p11 (about 2ms) at sp1 using spnam1=Sinc1.1000 and spoff1=0)
• Selective 90 ¹³C pulses applied on-resonance on the aliphatic ¹³C via the decoupler f2 channel (p13 (about 409u) at sp2, using spnam2=G4.256, spoff2=0).This same pulse is also applied to perform time reversed selective 90 pulses (p13 (about 409u) at sp8, using spnam8=G4tr.256, spoff8=0).
• Selective 180 ¹³C pulses applied on-resonance on the aliphatic ¹³C via the decoupler f2 channel (p14 (about 256u) at sp3, using spnam3=G3.256, spoff3=0).
• Selective 180 º ¹³C pulse applied off resonance on the CO resonances via the decoupler f2 channel (p14 (about 256u) at sp5, using spnam5=G3.256, spoff5=+120 ppm).
• Selective off-resonance CO decoupling via the decoupler f2 channel (pcpd2 at pl28=sp15 using cpdprg2=mlevsp180, spnam15=G3.256, and spoff15=+120ppm)
• 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  (31.8 ppm)	13CA+13CB (75 ppm)

 

Interpulse delays:

            d22 is optimized to 1/(4*J_N-CA) (12.4 ms)
            d23 is optimized to 1/(4*J_C-C) (3.5 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=8 and ds=16) 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 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 hncacb all command) to be used later.

Closely related pulse programs are also available:

• Gradient-Enhanced and ²H-decoupling (hncacbgp2h3d)
• Gradient-Enhanced using TROSY (trhncacbgp3d)
• Gradient-Enhanced and ²H-decoupling using TROSY (trhncacbgp2h3d)

List of Related Experiments:

Double-Resonance ¹H-¹³C experiments
Double-Resonance ¹H-¹⁵N experiments
Triple-Resonance ¹H-¹³C-¹⁵N backbone experiments
Triple-Resonance ¹H-¹³C-¹⁵N sidechain experiments