Splitting Pseudo-3D experiments into series of 2Ds in Topspin 3+

Interleaved experiments are easy to run as pseudo 3D experiments. A detailed method to design NMR experiments in Topspin 3+ is described previously. While running the experiments or after the run the user may have to split the data into their respective 2Ds to evaluate the results. This can be done easily by using “xfb” command along with right dimensions (13 or 23) as input in Topspin or NMRPipe processing scripts to process 3D datasets. Alternatively one might split these pseudo-3D data into individual 2Ds using Topspin 3+ macros. If the user has designed pseudo 3Ds with F2 dimension to be used to loop through the loop-counter values one could use these following lines as Topspin macro. Remember to place the macro in $PATH_TOPSPIN/exp/stan/nmr/au/src/user (The following script is called “splitrelax13”) int td, texpno=1000; GETCURDATA GETINT(“Enter the first target expno: “,texpno) FETCHPAR1S(“TD”,&td) i1=0; TIMES(td) RSER2D(13, i1+1,i1+texpno) i1 ++; END QUITMSG(“— splitrelax13 finished —“) This script will automatically read the number of 2D files to split the data into by reading the TD value and splitting the 3D data using RSER2D along 13 dimension starting from folder 1000 (user editable). Now if the interleaved experiment is looped through F1 dimension in Topspin aqpars, one will need to split the 3D along 23 dimension. This can be done in the following script. Remember to place the macro in $PATH_TOPSPIN/exp/stan/nmr/au/src/user (The following script is called “splitrelax23”) int td, texpno=1000; GETCURDATA GETINT(“Enter the first target expno: “,texpno) FETCHPAR3S(“TD”,&td) i1=0; TIMES(td) RSER2D(23, i1+1,i1+texpno) i1 ++; END QUITMSG(“— splitrelax23 finished —“) These scripts will create individual folders with 2D data in them which can be processed using standard 2D NMRPipe scripts or using “xfb” command in Topspin.

Designing interleaved experiment in Bruker Topspin 3+

Interleaved NMR experiments are often used to collect relaxation dispersion, zz-exchange, CPMG relaxation and T1/T2/NOE relaxation etc. This is often done to minimize the effect of degradation of sample, precipitation and aggregation over time on individual experiments. These aforementioned experiments can be done as a bunch of 2D experiments queued one after another, but the intensities of a spectra of slightly unstable protein could vary over time causing addition artifacts. Approach Design a working version of the 2D experiment First we need a 2D version of the experiment one wants to run where we create a segment in the pulse sequence that is varied between the different experiments. In the case of relaxation dispersion experiment this variable is referring to the number of CPMG pulses that are applied over a small mixing time of ~40ms and in the case of zz-exchange experiment, this variable refers to the time in milliseconds that is used to mix between two states under equilibrium. Let us try to use a complex case where the loop counter is varied where a counter is used to determine the number of times a segment is repeated. We first need to pass the value of –DCPMG via ZGOPTNS if we want to activate CPMG sections of the pulse sequence which can be done using “# ifdef CPMG” command as described below. # ifdef CPMG 5  d26                      ; tau_cp    (p61*2 ph3):N15         ;CPMG specific section    d26                      ; tau_cp    lo to 5 times l2 # endif This will iterate the three lines of the code “l2” number of times which is also supplied by user via Topspin. Now testing this pulse sequence is all that is left to confirm the correct implementation of the delays and pulses. Once this is conformed we would proceed to the following step where we collect thse experiments as a pseudo-3D experiment. Pseudo-3D experiment In an interleaved experiment, the first scan is done for first loop counter value, subsequent scans for the following loop counters and at the end of loop it should start from the first value all over again until the number of scans are exhausted. We would later split these individual experiments and combine with corresponding loop counter using a Topspin macro (discussed in another post). We will base this experiment on the 2D experiment where “l2” is used as a loop counter to vary the number of times a certain section of the code is run. The F1 and F2 dimension of the 2D experiment is going to be converted to F1 and F3 in our pseudo-3D experiment and F2 dimension will be used in the “QF” mode to vary the number of points in the loop counter. This has certain advantages over using F1 dimension as a loop counter as the chemical shift variable names, their corresponding increments and MC loop names have to be changed throughout the pulse sequence. With the use of F2 for loop counter eliminates this issue and one can keep the exact same variable names as […]