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    1 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
2 %                                                                             %
3 % Copyright (C) 2006,2008,2012-2014 Edward d'Auvergne                         %
4 %                                                                             %
5 % This file is part of the program relax (http://www.nmr-relax.com).          %
6 %                                                                             %
7 % This program is free software: you can redistribute it and/or modify        %
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15 % GNU General Public License for more details.                                %
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18 % along with this program.  If not, see <http://www.gnu.org/licenses/>.       %
19 %                                                                             %
20 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
21
22
23 % Calculating the NOE.
24 %%%%%%%%%%%%%%%%%%%%%%
25
26 \chapter{Calculating the NOE} \label{ch: NOE}
27 \index{NOE|textbf}
28
29
30 \begin{figure*}[h]
31   \includegraphics[width=5cm, bb=0 0 1701 1701]{graphics/analyses/noe_600x600}
32 \end{figure*}
33
34
35 % Introduction.
36 %%%%%%%%%%%%%%%
37
38 \section{Introduction to the steady-state NOE}
39
40 The calculation of NOE values is a straight forward and quick procedure which involves two components -- the calculation of the value itself and the calculation of the errors.
41 To understand the steps involved the execution of a sample NOE calculation script will be followed in detail.
42 Then the same operations will be presented for the perspective of the graphical user interface.
43
44
45
46 % From spectra to peak intensities.
47 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
48
49 \section{From spectra to peak intensities for the NOE}
50
51 For a set of recommendations for how to obtain the best quality relaxation rates, please see section~\ref{sect: spectra to intensities} on page~\pageref{sect: spectra to intensities}.
52 In summary the following are important -- temperature control (though the standard steady-state NOE single FID interleaved pulse sequences are fine), per-experiment temperature calibration, spectral processing with massive zero-filling and no baseplane rolling, and using an averaged peak list for determining the peak heights.
53
54
55
56 % Script UI.
57 %%%%%%%%%%%%
58
59 \section{Calculation of the NOE in the prompt/script UI mode}
60
61
62 % The sample script.
63 %~~~~~~~~~~~~~~~~~~~
64
65 \subsection{NOE script mode -- the sample script}
66
67 This sample script can be found in the \directory{sample\osus{}scripts} directory and will be used as the template for the next sections describing how to use relax.
68
69 \begin{lstlisting}
70 # Script for calculating NOEs.
71
72 # Create the data pipe.
73 pipe.create('NOE', 'noe')
74
75 # Load the sequence from a PDB file.
79
80 # Load the reference spectrum and saturated spectrum peak intensities.
83
84 # Set the spectrum types.
85 noe.spectrum_type('ref', 'ref_ave')
86 noe.spectrum_type('sat', 'sat_ave')
87
88 # Set the errors.
89 spectrum.baseplane_rmsd(error=3600, spectrum_id='ref_ave')
90 spectrum.baseplane_rmsd(error=3000, spectrum_id='sat_ave')
91
92 # Individual residue errors.
93 spectrum.baseplane_rmsd(error=122000, spectrum_type='ref', res_num=114)
94 spectrum.baseplane_rmsd(error=8500, spectrum_type='sat', res_num=114)
95
96 # Peak intensity error analysis.
97 spectrum.error_analysis()
98
99 # Deselect unresolved spins.
101
102 # Calculate the NOEs.
103 minimise.calculate()
104
105 # Save the NOEs.
106 value.write(param='noe', file='noe.out', force=True)
107
108 # Create Grace files.
109 grace.write(y_data_type='peak_intensity', file='intensities.agr', force=True)
110 grace.write(y_data_type='noe', file='noe.agr', force=True)
111
112 # View the Grace files.
113 grace.view(file='intensities.agr')
114 grace.view(file='noe.agr')
115
116 # Write the results.
117 results.write(file='results', dir=None, force=True)
118
119 # Save the program state.
120 state.save('save', force=True)
121 \end{lstlisting}
122
123
124
125 % Initialisation of the data pipe.
126 %~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
127
128 \subsection{NOE script mode -- initialisation of the data pipe} \label{NOE initialisation}
129
130 The start of this sample script is very similar to that of the relaxation curve-fitting calculation on page~\pageref{Rx initialisation}.
131 The command
132
133 \begin{lstlisting}[firstnumber=3]
134 # Create the data pipe.
135 pipe.create('NOE', 'noe')
136 \end{lstlisting}
137
138 initialises the data pipe labelled \promptstring{NOE}.
139 The data pipe type is set to the NOE calculation by the argument \promptstring{noe}.
140
141
142 % Spin systems.
143 %~~~~~~~~~~~~~~
144
145 \subsection{NOE script mode -- setting up the spin systems}
146
147 The backbone amide nitrogen sequence is extracted from a PDB\index{PDB} file using the same commands as the relaxation curve-fitting script (Chapter~\ref{ch: relax-fit}.
148 The command
149
150 \begin{lstlisting}[firstnumber=6]
151 # Load the sequence from a PDB file.
153 \end{lstlisting}
154 \index{PDB}
155
156 will load the PDB file \file{Ap4Aase\osus{}new\osus{}3.pdb} into relax.
157 Then the following commands will generate both the backbone amide and tryptophan indole $^{15}$N spins
158
159 \begin{lstlisting}[firstnumber=8]
162 \end{lstlisting}
163
164
166 %~~~~~~~~~~~~~~~~~~
167
169
170 The commands
171
172 \begin{lstlisting}[firstnumber=11]
173 # Load the reference spectrum and saturated spectrum peak intensities.
176 \end{lstlisting}
177
178 will load the peak heights\index{peak!height} of the reference and saturated NOE experiments (although the volume\index{peak!volume} could be used instead).
179 relax will automatically determine the format of the peak list.
180 Currently only Sparky\index{software!Sparky}, XEasy\index{software!XEasy}, NMRView\index{software!NMRView} and a generic columnar formatted text file are supported.
181
182 In this example, relax will determine from the file contents that these are Sparky\index{software!Sparky} peak lists (saved after typing \gui{lt}).
183 The first column of the file should be the Sparky assignment string and it is assumed that the 4$^{\textrm{th}}$ column contains either the peak height or peak volume (though this can be in any column -- the \prompt{int\_col} argument is used to specify where the data is).
184 Without specifying the \prompt{int\_method} argument, peak heights will be assumed.
185 See page~\pageref{uf: spectrum.read_intensities} for a description of all the \uf{spectrum\ufsep{}read\ufus{}intensities} user function arguments.
186 In this example, the peak list looks like:
187
188 {\footnotesize \begin{verbatim}
189      Assignment         w1         w2   Data Height
190
191         LEU3N-HN    122.454      8.397       129722
192         GLY4N-HN    111.999      8.719       422375
193         SER5N-HN    115.085      8.176       384180
194         MET6N-HN    120.934      8.812       272100
195         ASP7N-HN    122.394      8.750       174970
196         SER8N-HN    113.916      7.836       218762
197        GLU11N-HN    122.194      8.604        30412
198        GLY12N-HN    110.525      9.028        90144
199 \end{verbatim}}
200
201 For subsequent usage of the data in relax, assuming a 3D structure exists, it is currently advisable to use the same residue and atom numbering as found in the PDB file.
202
203 If you have any other format you would like read by relax please send an email to the relax development mailing list\index{mailing list!relax-devel} detailing the software used, the format of the file (specifically where the residue number and peak intensity\index{peak!intensity} are located), and possibly attaching an example of the file itself.
204
205
206
207 % Setting the errors.
208 %~~~~~~~~~~~~~~~~~~~~
209
210 \subsection{NOE script mode -- setting the errors}
211
212 In this example the errors where measured from the base plain noise.
213 The Sparky\index{software!Sparky} RMSD\index{RMSD} function was used to estimate the maximal noise levels across the spectrum in regions containing no peaks.
214 For the reference spectrum the RMSD was approximately 3600 whereas in the saturated spectrum the RMSD was 3000.
215 These errors are set by the commands
216
217 \begin{lstlisting}[firstnumber=19]
218 # Set the errors.
219 spectrum.baseplane_rmsd(error=3600, spectrum_id='ref_ave')
220 spectrum.baseplane_rmsd(error=3000, spectrum_id='sat_ave')
221 \end{lstlisting}
222
223 For the residue G114, the noise levels are significantly increased compared to the rest of the protein as the peak is located close to the water signal.
224 The higher errors for this residue are specified by the commands
225
226 \begin{lstlisting}[firstnumber=23]
227 # Individual residue errors.
228 spectrum.baseplane_rmsd(error=122000, spectrum_type='ref', res_num=114)
229 spectrum.baseplane_rmsd(error=8500, spectrum_type='sat', res_num=114)
230 \end{lstlisting}
231
232 There are many other ways of setting the errors, for example via spectrum duplication, triplication, etc.
233 See the documentation for the \uf{spectrum\ufsep{}error\ufus{}analysis} user function on page~\pageref{uf: spectrum.error_analysis} for all possible options.
234 This user function needs to be executed at this stage to correctly set up the errors for all spin systems:
235
236 \begin{lstlisting}[firstnumber=27]
237 # Peak intensity error analysis.
238 spectrum.error_analysis()
239 \end{lstlisting}
240
241
242 % Unresolved spins.
243 %~~~~~~~~~~~~~~~~~~
244
245 \subsection{NOE script mode -- unresolved spins}
246
247 As the peaks of certain spins overlap to such an extent that the heights or volumes cannot be resolved, a simple text file was created called \promptstring{unresolved} in which each line consists of the residue number followed by the atom name.
248 By using the command
249
250 \begin{lstlisting}[firstnumber=30]
251 # Deselect unresolved spins.
253 \end{lstlisting}
254
255 all spins in the file \promptstring{unresolved} are excluded from the analysis.
256
257
258
259 % The NOE.
260 %~~~~~~~~~
261
262 \subsection{NOE script mode -- the NOE calculation}
263
264 At this point the NOE can be calculated.
265 The user function
266
267 \begin{lstlisting}[firstnumber=33]
268 # Calculate the NOEs.
269 minimise.calculate()
270 \end{lstlisting}
271
272 will calculate both the NOE and the errors.
273 The NOE value will be calculated using the formula
274 \begin{equation}
275 NOE = \frac{I_{sat}}{I_{ref}},
276 \end{equation}
277
278 \noindent where $I_{sat}$ is the intensity of the peak in the saturated spectrum and $I_{ref}$ is that of the reference spectrum.
279 The error is calculated by
280 \begin{equation}
281 \sigma_{NOE} = \sqrt{\frac{(\sigma_{sat} \cdot I_{ref})^2 + (\sigma_{ref} \cdot I_{sat})^2}{I_{ref}}},
282 \end{equation}
283
284 \noindent where $\sigma_{sat}$ and $\sigma_{ref}$ are the peak intensity errors in the saturated and reference spectra respectively.
285 To create a file of the NOEs the command
286
287 \begin{lstlisting}[firstnumber=36]
288 # Save the NOEs.
289 value.write(param='noe', file='noe.out', force=True)
290 \end{lstlisting}
291
292 will create a file called \file{noe.out} with the NOE values and errors.
293 The force flag will cause any file with the same name to be overwritten.
294 An example of the format of \file{noe.out} is
295
296 {\scriptsize \begin{verbatim}
297 # mol_name          res_num  res_name  spin_num  spin_name  value                  error
298 Ap4Aase_new_3_mol1  1        GLY       1         N          None                   None
299 Ap4Aase_new_3_mol1  2        PRO       11        N          None                   None
300 Ap4Aase_new_3_mol1  3        LEU       28        N          None                   None
301 Ap4Aase_new_3_mol1  4        GLY       51        N          -0.038921946984531344  0.019031770246176943
302 Ap4Aase_new_3_mol1  5        SER       59        N          -0.312404225679127     0.018596937298386886
303 Ap4Aase_new_3_mol1  6        MET       71        N          -0.42850831873249773   0.02525856323041225
304 Ap4Aase_new_3_mol1  7        ASP       91        N          -0.5305492810313481    0.027990623144176396
305 Ap4Aase_new_3_mol1  8        SER       104       N          -0.5652842977581912    0.021706121467731133
306 Ap4Aase_new_3_mol1  9        PRO       116       N          None                   None
307 Ap4Aase_new_3_mol1  10       PRO       133       N          None                   None
308 Ap4Aase_new_3_mol1  11       GLU       150       N          None                   None
309 Ap4Aase_new_3_mol1  12       GLY       167       N          -0.7036626368123614    0.04681370194503697
310 Ap4Aase_new_3_mol1  13       TYR       175       N          -0.747464566367261     0.03594640051809186
311 Ap4Aase_new_3_mol1  14       ARG       200       N          -0.7524129557634996    0.04957018638401278
312 \end{verbatim}}
313
314
315
316 % Viewing the results.
317 %~~~~~~~~~~~~~~~~~~~~~
318
319 \begin{figure}
320   \centerline{
321     \includegraphics[
322       width=0.9\textwidth,
323       bb=0 -1 826 521
324     ]
325     {graphics/screenshots/noe_analysis/grace}
326   }
327   \caption[NOE plot]{
328     A Grace\index{software!Grace|textbf} plot of the NOE value and error against the residue number.
329     This is an example of the output of the user function \uf{grace\ufsep{}write}.
330   }
331   \label{fig: NOE plot}
332 \end{figure}
333
334
335 \subsection{NOE script mode -- viewing the results}
336
337 Any two dimensional data set can be plotted in relax in conjunction with the program \href{http://plasma-gate.weizmann.ac.il/Grace/}{Grace}\index{software!Grace|textbf}.
338 The program is also known as Xmgrace and was previously known as ACE/gr or Xmgr.
339 The highly flexible relax user function \uf{grace\ufsep{}write} is capable of producing 2D plots of any x-y data sets.
340 The two commands
341
342 \begin{lstlisting}[firstnumber=39]
343 # Create Grace files.
344 grace.write(y_data_type='peak_intensity', file='intensities.agr', force=True)
345 grace.write(y_data_type='noe', file='noe.agr', force=True)
346 \end{lstlisting}
347
348 will create one plot of the peak intensity of the reference and saturated spectra as different graph sets in the same plot as well as one plot for the NOE and its error.
349 The x-axis in all three defaults to the residue number.
350 Returning to the sample script three Grace data files are created \file{intensities.agr} and \file{noe.agr} and placed in the default directory \directory{.\ossep{}grace}.
351 These can be visualised by opening the file within Grace.
352 However relax will do that for you with the commands
353
354 \begin{lstlisting}[firstnumber=43]
355 # View the Grace files.
356 grace.view(file='intensities.agr')
357 grace.view(file='noe.agr')
358 \end{lstlisting}
359
360 An example of the output after modifying the axes is shown in figure~\ref{fig: NOE plot}.
361
362
363 % GUI.
364 %%%%%%
365
366 \newpage
367 \section{The NOE auto-analysis in the GUI}
368
370 This auto-analysis operates in the same way as the sample script described earlier in this chapter.
371 In this example, relax will be launched with:
372
373 \example{\\$ relax --log log --gui}
374
375 The \prompt{--log} command line argument will cause all of relax's text printouts to be placed into the \file{log} file which can serve as a record for later reference (the \prompt{--tee} command line argument could be used as well).
376
377
378 % Initialisation of the data pipe.
379 %~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
380
381 \subsection{NOE GUI mode -- initialisation of the data pipe}
382
383 First launch the analysis selection wizard (see Figure~\ref{fig: screenshot: analysis wizard} on page \pageref{fig: screenshot: analysis wizard}).
384 Select the NOE analysis and, if you plan on running steady-state NOE analyses from multiple fields in one relax instance, change the name of the analysis:
385
386 \begin{minipage}[h]{\linewidth}
387   \centerline{
388     \includegraphics[
389       width=0.8\textwidth,
390       bb=14 14 1415 1019
391     ]
392     {graphics/screenshots/noe_analysis/analysis_wizard1}
393   }
394 \end{minipage}
395
396 The second part of the wizard need not be modified, just click on \guibutton{Start} to begin.
397 This will create a dedicated data pipe for the analysis.
398 A data pipe bundle will also be created, but for the steady-state NOE will only contain a single data throughout the analysis.
399
400 \begin{minipage}[h]{\linewidth}
401   \centerline{
402     \includegraphics[
403       width=0.8\textwidth,
404       bb=14 14 1415 1019
405     ]
406     {graphics/screenshots/noe_analysis/analysis_wizard2}
407   }
408 \end{minipage}
409
410
411 % General setup.
412 %~~~~~~~~~~~~~~~
413
414 \subsection{NOE GUI mode -- general setup}
415
416 You should then see the blank analysis tab:
417
418 \begin{minipage}[h]{\linewidth}
419   \centerline{
420     \includegraphics[
421       width=0.8\textwidth,
422       bb=14 14 1415 1019
423     ]
424     {graphics/screenshots/noe_analysis/blank}
425   }
426 \end{minipage}
427
428 The first thing to do now is to set the NMR frequency label.
429 This is only used for the name of the NOE output file.
430 For example if you set the label to \guistring{500}, the file \file{noe.500.out} will be created at the end of the analysis.
431
432 You can also choose to change the \gui{Results directory} where all of the automatically created results files will be placed.
433 These two steps are unique to the GUI mode.
434
435
436 % Spin systems.
437 %~~~~~~~~~~~~~~
438
439 \subsection{NOE GUI mode -- setting up the spin systems}
440
441 Just as in the prompt and scripting UI modes, the molecule, residue and spin data structures need to be set up prior to the loading of any spin specific data.
442 The \gui{Spin systems} GUI element is used for this purpose.
443 Before any spin systems have been set up, this should say something like \gui{0 spins loaded and selected}.
444 To fix this, click on the \guibutton{Spin editor} button and you should then see the spin viewer window.
445 The next steps are fully described in section~\ref{sect: GUI - structural data} on page~\pageref{sect: GUI - structural data} for PDB files or section~\ref{sect: GUI - sequence file} on page~\pageref{sect: GUI - sequence file} for a sequence file.
446 The spin viewer window can now be closed.
447
448
449 % Unresolved spins.
450 %~~~~~~~~~~~~~~~~~~
451
452 \subsection{NOE GUI mode -- unresolved spins}
453
454 Using the unresolved spins file as described in the prompt/script UI sections, the same spins can be deselected at this point.
455 See Section~\ref{sect: GUI - deselect spins} on page~\pageref{sect: GUI - deselect spins} for the details of how to deselect the spins in the GUI.
456
457
459 %~~~~~~~~~~~~~~~~~~
460
462
463 The next step is to load the saturated and reference NOE peak lists.
464 From the main NOE auto-analysis tab, click on the \guibutton{Add} button in the \gui{Spectra list} GUI element.
466 From the first wizard page, select the peak list file containing the reference intensities (from the averaged shift list):
467
468 \begin{minipage}[h]{\linewidth}
469   \centerline{
470     \includegraphics[
471       width=0.8\textwidth,
472       bb=14 14 1415 1019
473     ]
474     {graphics/screenshots/noe_analysis/peak_intensity1}
475   }
476 \end{minipage}
477
478 Then set the obligatory spectrum ID string to a unique value (in this case \guistring{ref}).
479 The spectral dimension may need to be changed so that the peak intensities are associated with the correct atom of the pair.
480 In case you have forgotten the spin names or the format of the peak list next to the file name selection button is a preview button which can be used to open the peak list in the default text editor.
481 Set the other fields as needed.
482 Click on \guibutton{Next}
483 Note that a \prompt{RelaxWarning} will be thrown for all peak list entries which do not match a spin system within the relax data store.
484 This will cause the relax controller window to appear:
485
486 \begin{minipage}[h]{\linewidth}
487   \centerline{
488     \includegraphics[
489       width=0.8\textwidth,
490       bb=14 14 1415 1019
491     ]
492     {graphics/screenshots/noe_analysis/peak_intensity2}
493   }
494 \end{minipage}
495
496 Carefully check these warnings to be sure that the data is correctly loaded and, if everything is fine, the relax controller window can be closed.
497 If the dimension has been wrongly specified or some other setting is incorrect a \prompt{RelaxError} might appear saying that no data was loaded -- you will then need to fix the settings and click on \guibutton{Apply} again.
498 The error type page should now appear.
499
500 \begin{minipage}[h]{\linewidth}
501   \centerline{
502     \includegraphics[
503       width=0.8\textwidth,
504       bb=14 14 1415 1019
505     ]
506     {graphics/screenshots/noe_analysis/peak_intensity4}
507   }
508 \end{minipage}
509
510 Please read the description in this window very carefully to know what to do next.
511 In this example, we will choose \gui{Baseplane RMSD}.
512 For this specific example, Sparky's \guimenuitemthree{Extensions}{Spectrum}{Spectrum baseplane RMSD} option in the \gui{F1} selection mode was used to measure empty regions of the spectrum (mainly in the random coil region) to determine an average RMSD of approximately 3600.
513 Set the value and click on \guibutton{Apply}.
514
515 \begin{minipage}[h]{\linewidth}
516   \centerline{
517     \includegraphics[
518       width=0.8\textwidth,
519       bb=14 14 1415 1019
520     ]
521     {graphics/screenshots/noe_analysis/peak_intensity5}
522   }
523 \end{minipage}
524
525 As glycine 114 is located close to the noise signal, its error was much higher at 122000.
526 Individual spin errors can be set via the spin ID string (see section~\ref{sect: spin ID} on page~\pageref{sect: spin ID} for information about spin IDs):
527
528 \begin{minipage}[h]{\linewidth}
529   \centerline{
530     \includegraphics[
531       width=0.8\textwidth,
532       bb=14 14 1415 1019
533     ]
534     {graphics/screenshots/noe_analysis/peak_intensity6}
535   }
536 \end{minipage}
537
538 Finally select which type of spectrum this is and click on \guibutton{Finish}:
539
540 \begin{minipage}[h]{\linewidth}
541   \centerline{
542     \includegraphics[
543       width=0.8\textwidth,
544       bb=14 14 1415 1019
545     ]
546     {graphics/screenshots/noe_analysis/peak_intensity7}
547   }
548 \end{minipage}
549
550 The entire procedure should be repeated for the saturated spectrum (or you may have worked out that both can be loaded simultaneously by using the \guibutton{Apply} button more often).
551 For this example, the spectrum ID was set to \guistring{sat} and the baseplane RMSD to 3000 for all spins (except for G114 which had an error of 8500).
552
553 The NOE analysis tab should now look like:
554
555 \begin{minipage}[h]{\linewidth}
556   \centerline{
557     \includegraphics[
558       width=0.8\textwidth,
559       bb=14 14 1415 1019
560     ]
561     {graphics/screenshots/noe_analysis/analysis_tab2}
562   }
563 \end{minipage}
564
565
566 % The NOE.
567 %~~~~~~~~~
568
569 \subsection{NOE GUI mode -- the NOE calculation}
570
571 Now that everything is set up, simply click on \guibutton{Execute relax} in the NOE analysis tab.
572 The relax controller window will appear displaying many messages.
573 These should all be checked very carefully to make sure that everything has executed as you expected.
574 The \gui{Results viewer} window will also appear:
575
576 \begin{minipage}[h]{\linewidth}
577   \centerline{
578     \includegraphics[
579       width=0.8\textwidth,
580       bb=14 14 1415 1019
581     ]
582     {graphics/screenshots/noe_analysis/fin}
583   }
584 \end{minipage}
585
586 The results viewer window can be used to launch a text editor to see the NOE values and error or Grace to visualise the results (see Figure~\ref{fig: NOE plot} on page~\pageref{fig: NOE plot}).
587
588 As a last step, the relax state can be saved (via the \guimenuitemone{File} menu) and relax closed.
589 Take one last look at the \file{noe.out} log file to be certain that there are no strange warnings or errors.