S³(H^N) TROSY ^hHNCO

Ref.:
A.Meissner, and O.W.Sørensen J.Magn.Reson. 143, 387-390 (2000). (New Techniques for the Measurement of C'N and C'H^N J Coupling Constants across Hydrogen Bonds in Proteins)

Delays: = (2J_N,H)^-1; T = n(¹J_N,CO)^-1, n = 1,2; T' = T - (duration of water-selective /2 pulse);  = gradient delay.

Phases for Varian Unity Inova:  = -y; {_I = y; _S = y} (echo) and {_I = -y; _S = -y} (antiecho).

Phases for Bruker DRX:  = y; {_I = -y; _S = -y} (echo) and {_I = y; _S = y} (antiecho).

For measurement of ^3hJ(N,CO) by quantitative approach set flag
select = 'a' for left line spectrum
         'c' for left line reference spectrum

Recommendation for simultaneous measurement of ^2hJ(H^N,CO) and ^3hJ(N,CO): run select='a','b' with a large number of scans followed by a short select = 'c' experiment.

Set array = 'phase2,phase,select' for recording interleaved data.

For 2D sequence without C pulse set ni2 = 0 for 2D sequence with C pulse set ni2 = 1.

Set dof to the middle of the CO region. For adjustment of other parameters, please refer to the comments in the header of the pulse sequence.

To generate the selective pulse shapes, copy water_sc.inp, ib_co_onr.inp, ib_ca_off.inp, and wurst2_ca.inp to your $HOME/vnmrsys/shapelib directory, adjust ref_pwr and ref_pw90 according to your proton (water_sc.inp) and carbon (ib_co_onr.inp, ib_ca_off.inp, and wurst2_ca.inp) pulse calibration. Adjust the offset for the C selective shapes (ib_ca_off.inp, and wurst2_ca.inp) to -120*dfrq. Type the command: Pbox -f >filename<.inp for each of the files to generate the shapes.

An output like
>>> Set pulse width to 1.0000 ms <<<
>>> Set pulse power to 21 dB <<<
appears.

Remember to adjust the parameters according to the output. Set the power level for Wurst-2 decoupling (Cdecpwr) 2 dB higher than output.

Processing of interleaved 3D S³(H^N) TROSY ^hHNCO data:

Sorting of datasets recorded with array = 'phase2,phase,select'

• change into the directory of your data set (cd name.fid)
• rename the fid file >mv fid fid_orig<
• run the sort_3d sorting routine >sort_3d fid_orig fid ni ni2 np dim< (dim = dimension of select array: select = 'a','b' -> dim = 2 / select = 'a','b','c' -> select = 3)
• move the new fid files to different directories >mv fid_# ../new_file_#.fid/fid< (# = integer number)
• copy the procpar file to the new directories >cp procpar ../ new_file_#.fid< (# = integer number)
• use your usual software for processing the subspectra. The same phase correction should be applied in all cases.

sort_3d.c installation:
change to the directory with the sort_3d.c file
type cc sort_3d.c -o $HOME/bin/sort_3d
this will compile the program and save the executable file in the $HOME/bin directory. Make sure the directory exists or choose another location. Make also sure your PATH environment variable contains the directory you are using.
 
 

Content
Pulse sequence:   hbond_hc.c
Parameters:       hbond_hc.par
Pbox input files: water_sc.inp
                  ib_co_onr.inp
                  ib_ca_off.inp
                  wurst2_ca.inp
Sorting routine:  sort_3d.c
 

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