Hnco-ge-relax fully relaxed accordion HNCO (Tjandra 2009)

The pulse sequence is based on a 3D HNCO experiment, where indirect amide ¹⁵N and carbonyl ¹³C dimensions are recorded in constant time mode. The key modification is adding T1 and T2 relaxation loops to the sequence prior to the indirect evolution delays.

In the constant time evolution experiments, indirect dimension FIDs have periodic profile with no decay. However this pulse sequence introduces decay to those profiles, corresponding to T1 and T2 relaxation processes, therefore relaxation rates can be extracted directly from the line width after the fourier transformation.

Length of the relaxation delay is incremented simultaneously with the corresponding (indirect dimension) evolution delay, i.e. in the accordion fashion. Therefore, the amount of relaxation in a given indirect FID is proportional to the indirect evolution time, yielding the exponentially decaying profile.

The chemical shift and relaxation evolve at different time scales, because the corresponding time increments are not equal. So in order to extract the relaxation time, a scaling factor between the two increments needs to be taken into account.

Relaxation time T_r can be calculated as:

Here , and is the relaxation delay increment.

Section on dimensions explains how to calculate the scaling factor .

The two indirect dimensions in this experiment simultaneously record chemical shift and relaxation rates. The last section below explains how to calculate the scaling factors.

Spectral window and relaxation delay increment are hardcoded in both dimensions.

δN (amide),T2 or T1 dimension

• in22=in24=in25=1/(4*swN), where swN - spectral window in ¹⁵N dimension
• T2 loop increment : 24*d13 (12 ms)
• loop counter NLOOP - hardcoded number of complex points
• #define T2 turns on T2 loop
• #define T1_onN switches this dimension to record T1 relaxation
• detection mode: Echo-Antiecho

δC',T1 dimension

• in0=in10=1/(4*swC'), where swC' - spectral window in C' dimension
• detection mode: States-TPPI

Calculation of scaling factors

Scaling factors are calculated as follows:

factor	formula	default value
		21.43
if #define T1_onN is uncommented and #define T2 is commented out		57.14
		50

• definition: channel H
• carrier: ~ 4.8 ppm (H₂O)
• p1 @ pl1 - high power 90^{o 1}H pulse
• decoupling cpds1: p30 @ pl30 - 90^o pulse (60 μs for 600 MHz)

Two ¹³C channels are used in the sequence: one for Cα and one for C' (carbonyl). Channel definitions are set in file bits.nt. You might have to tweak that file and the frequency offsets for the shaped pulses if you have only one channel for ¹³C.

Cα

• definition: channel C1
• carrier: ~56 ppm (center of Cα region)
• p4 @ pl4 - soft 180^o Cα pulse (with null @ CO) 47.4 μs for 600 MHz

C'

• definition: channel C2
• carrier: ~177 ppm (center of C' - carbonyl - region)
• shaped pulse sp1: p6, sinc. 180^o pulse on C', 191.7 μs for 600 MHz
• shaped pulse sp2: p6, sinc. 90^o pulse on C', 191.7 μs for 600 MHz

• definition: channel N
• carrier: ~115 ppm (center of amide nitrogen region)
• p7 @ pl7 - high power 90^{o 15}N pulse
• decoupling cpd2: p31 @ pl31 - soft 90^{o 15}N pulse for waltz16, 190 μs for 600 MHz

Durations of gradient pulses are hard-coded in the program

All gradients are sine-shaped, except gp24-27, which are square.

Tune p26 or p24/p25, p24+p25=p26.

• p19:gp19, 20% on X
• p21:gp21, 25% on Y
• p22:gp22, 30% on X
• p23:gp23, 30% on Y
• p24:gp24, square 30% on Z
• p25:gp25, square -30% on Z
• p26:gp26, square -30% on Z
• p26:gp27, square 30% on Z
• p29:gp29, 30% on Y

• #define HEAT - turns on sample heating compensation loop
• #define NITRO - ???

1. Chen, K and Tjandra, N. Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment. Journal of Magnetic Resonance , 2008. BibTeX [chen08]