1) Do all the xfb transformations using the NC_proc argument with the same parameter. For example, you can do always "xfb NC_proc 0". This keeps the program from applying its own rescaling to the data, so you don't lose information about relative intensity among spectra. If you don't do this, you get crazy NOEs and unfitable T1 and T2 data.
2) Be sure that you apply the same phase and baseline parameters to all the T1 experiments. The same applies to T2 and NOE.
NOTE ON PROCESSING THE NOE EXPERIMENT:
The Heteronuclear NOE experiment is acquired in the interleaved mode, this means that the irradiated and non-irradiated spectra are acquired as a single experiment, each scan alternating the irradiation on and off. In order to process this spectrum you need to use an AU program called "splitinvnoe".
When you're done, copy all the peaks from the spectrum where you tuned the positions (command "oc") and paste them to all other sectra ("op").
Now go back to the spectrum where you can see nice peaks, select all the peaks, and type "rh". This opens the "Relaxation Heights" window. Select "Heights at each position in each spectrum", enter "Setup" and in the setup window chose which spectra you want to include in the analysis (typically all) and enter their corresponding time delays in ms. Accept this window, also accept the "Relaxation Heights" window, and type "rh" again: the "Relaxation Heights" window will appear, this time with a table of time constants, their standard deviations, and the heights taken from each spectrum. This table can be saved in text format so as to be imported in a spreadsheet. Besides, if you click on a row you can see the decay profile for the corresponding aminoacid.
For each spectrum, do "lt" and save the peak list including height information. Import both files in a spreadsheet, and calculate the NOE as the ratio between the intensities of the irradiated and non-irradiated spectra.
Prepare a spreadsheet with this columns:
A= AA number | B= T1(s) | C= T2(s) | D= (T1-T1av)/T1av | E= (T2-T2av)/T2av | F = D-E
Where T1av and T2av are the average T1 and T2 values repectively (that is, average of columns B and C)
Now create a column G whose value will be:
- "OK" if the absolute value of F is smaller than 1.5 times the standard deviation of column F
- "no!!!" otherwise
In Excel this could be done like this (example for row 3, spanish version):
=SI(ABS(F3)>DESVEST(F)*1.5,"","OK")
All those aminoacids for which column F is "OK" can be used for analysis of its R2/R1 ratio if the NOE criterium is also "OK" (see below).
Now put in column H all the NOE values, and create column I, which will be:
- "OK" if the NOE is bigger or equal than 0.6
- "no" otherwise
In Excel (example for row 3, spanish version):
=SI(H3<0.6,"OK","no!!!")
Now calculate, in column J, the R2/R1 ratio (=T1/T2) for those aminoacids who have "OK" in columns F and H. Calculate the average value and standard deviation of this ratio, and write down these numbers (they will be used in the tmest program)
Run the tmest program and answer its questions:
Enter "15n" to the "input nucleous" question
"600.13" for the "proton spectrometer field"
The average and standard deviation in the R2/R1 ratio, separated by a comma
An initial guess for the tau-m (in nanoseconds)
Prepare a spreadsheet with this columns (you can use this template):
A= AA number | B= R1(1/s) | C= error in R1 | D= R2(1/s) | E= error in R2 | F = NOE | G= error in NOE
Column I will be J(wH) and J its error:
I = (F-1)*(B/5)*(-0.101)*4/72100^2
J = RAIZ((B*G)^2+(C*(F-1))^2)*(0.101*4*0.2/72100^2)
Column K will be J(wN) and L its error:
K = 4 /(3*72100^2+4*35400^2)*B*(1-(7/5)*(-0.101)*(F-1))
L = 4 /(3*72100^2+4*35400^2)*RAIZ(((1-(7/5)*(F-1)*(-0.101))*C)^2+((-7/5)*(-0.101)*G)^2)
Column M will be J(0) and N its error:
M = 1 /(3*72100^2+4*35400^2)*(6*D-3*B-(18/5)*B*(F-1)*(-0.101))
N = 1 /(3*72100^2+4*35400^2)*RAIZ((6*E)^2+(-3*C+(18/5*0.101)*(C*(F-1)))^2+((18/5*0.101)*(B*F))^2)