Using Assignment

Two crucial steps in structure determination using NMR spectra are to assign each nucleus to a specific chemical shift (so-called sequence-specific assignment) and to assign each peak in relevant spectra to these assigned resonances. These two steps together constitute the assignment procedure. Three example lessons are presented in the following section, which highlight the basic steps.


Lesson 1: Homonuclear 2D assignment strategy

This lesson presents the basic steps of an NMR spectrum assignment using homonuclear spectra, including TOCSY, DQF-COSY, and NOESY 2D NMR spectra of Zn-rubredoxin (Blake et al. 1991).

The topics covered in this lesson are:

1.   Setting up for the lesson

If not done yet, set up the tutorial files as described in "Setting up tutorial files" in the preface, How to use this book. The files for this lesson are located in the Assignment\Lesson1 folder.

2.   Starting FELIX

Start FELIX by double-clicking the FELIX icon on your desktop, or by clicking the Start button on the Windows taskbar, then selecting Programs/Accelrys FELIX 2004/FELIX 2004. If FELIX prompts you to restore from last session, click Cancel.

Change your Current Working Directory to C:\Felix_Practice\Assign\Lesson1\ using the Preference/Directory... command. Build a new database by opening the File/New... command and choosing Create a new matrix or DBA file. Make sure the File Type is set to DBA(*.dba). Enter zn.dba and click OK.

3.   Going to the Assign module

Select the Assign/Project item from the menubar.

4.   Setting up the database

When FELIX informs you that no project was found, click OK. FELIX will prompt you for a new project.

In the control panel, the default name of the project appears as asg:project. You can enter another name if you want (e.g., zn:project).

In the next control panel, select the linear chain of the molecule by setting File name to znrdlec.car.

Click OK to build the entities and read in the molecule.

This procedure typically takes several seconds. Then the program asks for the library. The library is an ASCII file, as described in "Assign/ Define Library" in the separate FELIX User Guide. FELIX contains a standard library for proteins and DNA (pd.rdb) which you should read in.

In the next control panel, select the Define Library from File option and click OK.

In the following control panel, select pd.rdb.

This is the protein/DNA library. A few seconds later the project setup procedure finishes.

5.   Viewing the project entity through a spreadsheet

Select the Assign/Project menu item again.

The project entity is presented in the spreadsheet, and you can browse through its fields.

Many fields contain zeros or nulls, since the full definition is not finished yet. There are nine experiment columns, therefore you can define nine experiments in one project.

Select File/Close in the table to exit the spreadsheet.

6.   Adding an experiment to the projects

Select the Assign/Experiment menu item to define new experiments in the assignment database. Select zc.mat (the DQF-COSY spectrum).

Click OK.

Set the following parameter values for the plot using the 2D Display Parameters control panel:

Contour Threshold 0.005
Color Scheme Blue/Green
Axis type D1 ppm
Axis type D2 ppm

Leave the other parameters at their default values and select Apply.

Click OK when the message box appears.

If you want, you can change the display parameters using the Experiment/Change Attribute menu item in the Experiments table.

The program plots a density or contour plot of the DQF-COSY using the parameters you defined. The coloring scheme is a predefined blue and green colormap with 16 blue colors for positive peaks and 16 green colors for negative peaks.

What you enter for Experiment Title should be descriptive, but not too long (for example, COSY or DQF is appropriate for this spectrum). Leave Use Default Names toggled on (which automatically fills in the peak table, volume table, and J table names). Set the remaining parameters to these values:

Type 2D DQF
Temperature 298
pH 7
Solvent Water
D1 Nucleus Proton
D2 Nucleus Proton
D1 Tol 0.008
D2 Tol 0.008
W1 D2
W2 D1
W1-W2 Transfer J-coupled
# of J Steps 3

Click OK.

It is important to define the spectrum-specific tolerances, which are used in many automated and semi-automated procedures.

7.   Repeating Step 6 for the TOCSY and NOESY spectra

Select the Project/Experiment menu item again.

This brings up a spreadsheet with the currently-defined experiments. You can use this spreadsheet to add, delete, or edit experiments.

Now go to the spreadsheet menubar and select the Experiment/Add item.

When the control panel appears, select zh.mat for TOCSY and zn.mat for NOESY. The required values for each run are different:

Parameter Name TOCSY Value NOESY Value
Contour Threshold 0.015 0.01
Color Scheme Magenta Cyan
Number Of Levels 10 10
Negative Levels Off Off
Axis Type D1 ppm ppm
Axis Type D2 ppm ppm

Leave the remaining parameters at their default values and click OK.

In the next control panel, set these parameter values for TOCSY:

Parameter Name TOCSY Value NOESY Value
Experiment Title tocsy noe
Use Default Name on on
Type 2D TOCSY 2D NOESY
D1 Nucleus Proton Proton
D2 Nucleus Proton Proton
W1 D2 D2
W2 D1 D1
W1-W2 Transfer J-coupled NOE
Number of J Steps 7 0
Mixing Time 0 0.05
D1 Tolerance 0.008 0.01
D2 Tolerance 0.008 0.01

Leave the remaining parameters set to their default values.

8.   Checking the project entity

You may need to highlight the spectral window if the main menu is not displayed. Next select Assign/Project to display the Project table. You can click the maximize button in the upper right corner of the table to expand it for a better view. After reviewing the items in the table, select File/Close to close it.

Note that previously zero or null fields now have values.

9.   Drawing the full DQF-COSY spectrum

If necessary, select Window/Auto Arrange to re-arrange the Experiment table and the spectral window.

Now go to the Experiments table and select the cosy spectrum by clicking the first row and then clicking the Draw icon.

The next step in the assignment procedure is to do a peak picking. This procedure is very important, since all other steps rely on proper peak picking.

Usually peak picking involves several steps. First the automatic peak picker should be run. You can run the regular peak picker or the Stella peak picker. The results are then filtered automatically (symmetrizing, deleting the diagonals, deleting artifacts (solvent ridges), and deleting peaks with invalid widths). You should also thoroughly inspect the results visually, to ensure there is enough confidence in the data. FELIX also provides a tool to fit the 2D peaks via the Peaks/Optimize menu item (see "Peaks/Optimize" in the separate FELIX User Guide), which also increases the accuracy of peak picking.

Note: The importance of peak picking cannot be overemphasized, since the automated assignment tools work only as well as the starting conditions permit ("garbage in garbage out"). Bearing this in mind, Accelrys has tried to give you a clean peak set. Therefore you need to read this peak set from the text directory provided (dqf.xpk, tocsy.xpk and noe.xpk).

10.   Reading in the peaks

From the main menu, select the File/Import/Peaks menu item. Set the parameter Peak File Type to FELIX Peak File(*.*). Select dqf.xpk as File name. Leave FELIX Peak Table Name at its current setting (xpk:cosy), since your current experiment is a DQF-COSY.

Click OK.

When the query box appears, asking about overwriting the entity, select Overwrite.

The footprints of the imported peaks are displayed on the DQF-COSY spectrum. You may need to click the Plot icon from the main tool bar to redraw the spectrum. A Peaks-xpk:dqf table is also displayed, showing the imported peaks.

Go to the Experiment table and select the TOCSY spectrum. Click the Draw icon to display it.

From the main menu select the File/Import/Peaks menu item. Set the Peak File Type to FELIX Peak Type(*.*). Select tocsy.xpk as File name and click OK.

When the query box appears, asking about overwriting the entity, select Overwrite.

Select File/Close from the Peaks-xpk:tocsy table menu to close the table.

Next you repeat the procedure for the NOE spectrum.

Select the NOE spectrum in the Experiments table and click the Draw icon to display it.

From the main menu, select the File/Import/Peaks menu item. Set the Peak File Type to FELIX Peak Type(*.*). Select noe.xpk as File name. Leave FELIX Peak Table Name as xpk:noe and click OK.

When the query box appears, asking about overwriting the entity, enter Overwrite.

Select File/Close from the Peaks-xpk:noe table menu to close the table.

Now you have a full peak set defined for all three experiments.

11.   Selecting the DQF-COSY spectrum

Select the DQF-COSY spectrum in the Experiments table and click the Draw icon. Highlight the spectral window and if necessary, press <Ctrl>-f on your keyboard to obtain the full plot.

The displayed footprints belong to this spectrum, not to the NOESY, which was read in last. The database took care of reloading the spectrum-specific information.

The next step is the collection of prototype patterns, i.e., sets of frequencies, which later are promoted to patterns and assigned to specific amino acid residues. The menu items relating to prototype patterns are in the third subsection of the Assign pulldown. First we demonstrate a method which uses all three available (COSY, TOCSY, and NOESY) spectra to generate prototype patterns.

12.   Performing a prototype pattern detection

Select the Assign/Collect Prototype Patterns menu item. From the control panel select the 2D Homonuclear option and click OK.

In this tutorial the homonuclear 2D spectra are used for assignment.

In the subsequent control panel, set Spin System Type to Proteins and Systematic Search to Method. Click OK.

A control panel with several options appears. The program tries to fill in reasonable values.

.

Set these parameter values in the control panel:

Method: COSY+TOCSY+NOE
COSY experiment: cosy
TOCSY experiment: tocsy
NOE experiment: noe
Seed/Expansion: Use Defaults
Frequency Collapse Tolerance: 0.015
Output level: Low

The Frequency Collapse Tolerance is the tolerance for aligning and finding connected expansion peaks with seed peaks.

At this point you could just start the collection and use the defaults for the seed peak and expansion peak area. You can also look at them by clicking the More... button instead of the OK button. In the next control panel leave all parameters at the default values:

Seed area D1
Low 6
High 12
Seed area D2
Low 3
High 5.5
Expansion area D1
Low -2
High 12
Expansion area D2
Low -2
High 12
Remove Intraproto Frequencies on Number of Frequencies in Proto
Min 3
Max 8
Number of Iterations 6
Frequencies Per Iteration 1

The Seed Area D2 (High) is the amide proton region above the diagonal. Remove Intraproto Frequency on Number of Frequencies in Proto is the minimum and maximum number of frequencies in a prototype pattern. Number of Iterations is the maximum number of expansion loops, and Frequencies Per Iteration is the number of frequencies in each loop to keep.

In the remaining part of the control panel, set these parameter values:

# ppm filters active 2
Filter #
1
Low
6
High 12
Min 1
Max 1
2
Low 3
High 5.5
Min 1
Max 3

Click OK.

Only those prototype patterns are kept which have at least (and at most) one frequency in the 6-12 ppm region (amide proton) and at least one (and at most three) frequencies in the 3-5.5 ppm region.

Be sure to leave the Min # cont (the minimum number of contacts) values at their defaults (1 1 2 2, 1 2 2 3, and 2 2 3 4).

In the output window, information is displayed about the current stage of prototype pattern collection. After one minute, the prototype pattern collection is finished for 106 seed peaks and 3240 expansion peaks, and the following information appears in the output window:

     Nr of prototype patterns generated:(57) 
The 2D protopattern detection took 7 seconds

Also, a spreadsheet containing the prototype patterns is displayed (Protopatterns).

13.   Saving the results of prototype pattern detection as a file

Highlight the Protopatterns table and select the File/Save As menu item. Set the File name as zn_protos.txt and click OK.

In the output window you are informed about the success of the command:

Saved table:table 'Protopatterns' (entity zn:proto)as File
'C:\tutorial\Assignment\Lesson1\zn_protos.txt'.

The next step is to visually inspect the prototype patterns. The Protopatterns spreadsheet provides several ways for you to see prototype patterns: you can draw frequencies of prototype patterns as lines on top of a contour plot, spawn tiles, or draw a strip plot.

Go to the Protopatterns table and select the Preferences/Draw menu item.

When the control panel appears, set Vertical Color to Blue and Horizontal Color to Green. Click OK.

Now click the first row of the table (select the first prototype pattern) and click the Draw icon.

You see four lines at 9.7, 5.37, 1.78, and 0.89 ppm, which are frequencies in this prototype pattern.

Right-click in the spectral window and select the Clear Frequencies command in the context menu to clean the frequency lines.

The second way to visualize prototype patterns is to spawn tile plots from them. This allows you to concentrate only on frequencies and peaks belonging to them, which are present in this prototype pattern.

14.   Making a tile plot of prototype pattern 1

Reselect the first prototype pattern from the table and click the Tile Plot icon. If you want to change the tile plot attributes, go to the table and select the Preferences/Tile Plot menu item.

The tile plot is displayed.

Highlight the spectral window if necessary, and press <Ctrl>-c (if you were in intensity-plot mode) to see the contour plot of the COSY spectrum tiled by the first prototype pattern.

Select the tocsy spectrum from the Experiments table and click Draw.

Using the tile plot functionality, you can concentrate on peaks and their immediate surroundings which belong to a prototype pattern. Also, you can use strip plots to see strips surrounding the frequencies in vertical or in horizontal position.

15.   Returning from tile mode

While the spectral window is highlighted, press <5> in the keypad and use the large cross-hair cursor to pick one of the boxes. This command (Jump) places only that small region on the screen and exits tile-plot mode.

Go to the Protopatterns table and select the Preferences/Strip Plot menu item. Set these parameters:

Shift Type Generic
Dimension W2
Width 64
Scale 4.

Click OK.

Click the Strip Plot icon in the table.

You see four vertical strips with the frequencies of the first prototype pattern in the middle of each.

From the strip plot you can see that there are no outstanding peaks that have common chemical shifts with the frequencies in this prototype pattern. Therefore you can continue to promote this prototype pattern to pattern. The first step in this procedure is to copy these frequencies to the clipboard.

16.   Copying a prototype pattern to the frequency clipboard

While the spectral window is highlighted, select the Assign/Frequency Clipboard/Copy Proto to Clipboard menu item. In the control panel, select 1 as the Proto Pattern and click OK.

The first prototype pattern is now copied to the clipboard list. This list can be manipulated (you may add or delete frequencies to or from the list, swap the order of two frequencies, delete duplicate frequencies, sort the list, or zero the list). You can also display the list as lines on top of the matrix plot or spawn a tile and strip plot from it.

Select the Assign/Frequency Clipboard/Sort Clipboard menu item. Now you can sort the frequencies in the clipboard in descending ppm order by toggling Descending order to on.

Next select the Assign/Frequency Clipboard/View Clipboard menu item to view the content in the clipboard.

The results should look like this:

The Frequency Clipboard List contains the following frequencies:
     # Freq(ppm) Atom

--- --------- ----

1 9.703 X
2 5.369 X
3 1.786 X
4 0.895 X

If there is no appropriate frequency to add or delete, the clipboard list can be promoted to a pattern, and the pattern can be then subjected to database searches and naming.

17.   Copying the frequency clipboard to the pattern

Select the Assign/Frequency Clipboard /Copy Clipboard to Pattern menu item. In the control panel, keep all the default settings and click OK.

Now the four frequencies in the clipboard are promoted to a spin system. Also, a new spreadsheet is displayed - Spinsystems. You can list this pattern to a file or to the output window or you can examine it in the spreadsheet. Also, you can close this spreadsheet and reopen it using the Edit/Spin Systems menu item.

18.   Viewing the pattern

While the spectral window is activated, select the Assign/Report Spin System menu item. Make sure that Action is set to To Textport and click OK.

This prints the following information to the output window:

Listing for pattern : pa1
comment : null
color : Red
root frequency : 9.703

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
cosy tocsy noe null null null null null null
9.703 9.703 9.703 9.703 9.703 9.703 9.703 9.703 9.703 9.703 1:*_*:HX
5.369 5.369 5.369 5.369 5.369 5.369 5.369 5.369 5.369 5.369 1:*_*:HX
1.786 1.786 1.786 1.786 1.786 1.786 1.786 1.786 1.786 1.786 1:*_*:HX
0.895 0.895 0.895 0.895 0.895 0.895 0.895 0.895 0.895 0.895 1:*_*:HX

The neighbor probabilities
--------------------------
Pattern Probability

The residue type probabilities
------------------------------
Residue Probability

Next you copy the generic shifts for the frequencies to the spectrum-specific category. Since these chemical shifts were detected in the TOCSY spectrum, you can copy this to the experiment without any change.

19.   Finding the residues that the pattern belongs to

Select the Assign/Residue Type/Score Residue Type menu item. Toggle off Use All Patterns. Select the pa1 pattern, set the Max Std Dev to 3, and set the Min Atoms to Specify and 4. Set the Scoring method to All Atoms, and Database to Store. Click OK.

In the output window, a report is generated of the probabilities that this pattern belongs to a certain type of amino acid residue:

Scoring pattern pa1
9.703 5.369 1.786 0.895
`H' `H' `H' `H'
... vs ile | score=1 | aver.=1.249352 | matched atoms=4 / 4
... vs leu | score=1 | aver.=2.018772 | matched atoms=4 / 4
... vs lys | score=1 | aver.=1.940873 | matched atoms=4 / 4
... vs thr | score=0.8 | aver.=1.6740694 | matched atoms=4 / 5
... vs val | score=1 | aver.=1.569354 | matched atoms=4 / 4
Scoring is done.

Since the highest score and lowest average is for the Ile and Val residues, since you have seen from strip plots that there are no extra resonances, and since Ile theoretically has seven resonances, while Val has only five (with methyl degeneracy likely four), you can now assume that pattern pa1 is a valine type.

Generally, the probability is higher if the score higher and the average is lower. The best-matched atoms give a higher confidence in the probability. You can store the result with the same control panel, by selecting the Store Result option.

You can try to perform this action again, using the DQF-COSY peaks to help distinguish between equally likely residue types. If you do, the printout will be:

Scoring pattern pa1
9.703 5.369 1.786 0.895
'H' 'H' 'H' 'H'
... vs ile | score=0.375 | aver.=1.249352 | matched atoms=4 / 4 | cosy=3 / 8
... vs leu | score=0.25 | aver.=2.018772 | matched atoms=4 / 4 | cosy=2 / 8
... vs lys | score=0.0526316 | aver.=1.940873 | matched atoms=4 / 4 | cosy=1 / 19
... vs thr | score=0 | aver.=1.670694 | matched atoms=4 / 5 | cosy=0 / 3
... vs val | score=0.75 | aver.=1.569354 | matched atoms=4 / 4 | cosy=3 / 4
Scoring is done.

This can help in further distinguishing residue types. After you decide that this pattern is a valine type, you need to see which frequency belongs to which atom. For this you must query the database.

20.   Querying the database

Select the Assign/Residue Type/Match Residue Type menu item. Select pa1 as the pattern and select the Val residue to match against it. Click OK.

The result is a table in the output window showing the relative differences of each frequency from its expectation value. The smallest absolute value shows the highest matching:

     Matching pattern pa1 versus val
9.703 5.369 1.786 0.895
H H H H
HN 2.579 -4.526 -10.400 -11.861
HA 10.187 2.307 -4.207 -5.827
HB 30.572 13.236 -1.096 -4.660
HG1* 40.332 20.632 4.345 0.295
HG2* 48.906 24.828 4.922 -0.028

You can see that the frequency with 9.703 ppm probably belongs to the HN resonance and that the Ha is the frequency with 5.369 ppm. Hb is the frequency with 1.786 ppm, The two gamma methyls are not resolved, but the 0.895 ppm frequency belongs to them. You need to set these findings in your database. To do this, you must assign these frequencies.

21.   Assigning the frequencies

Select the Assign/Assign Spin System/Frequency menu item. Select pa1, and click Next. This brings up a control panel in which you can make the assignment. Choose the first frequency 9.703 in the Frequency list and click the Select button. Select the VAL item from the Residues list and click the Filter button. This fills in the #S list with 4, 37, and *.

Since you do not know which valine this pattern belongs to, choose the wild card (*), then select the HN item from the Nuclei list. Click the Build button, which fills in the Atom Spec with 1:VAL_*:HN. Now click Add. Since the database contains only atoms that belong to certain residues and the selected residue is not a real one, a dialog box appears asking you for confirmation to include this "new atom" in the database. Click Yes.

Now repeat the procedure for the other frequencies in this pattern. When you are finished, select Quit.

You can see the results of this assignment by using the Assign/Report Spin System menu item.

LISTING FOR PATTERN pa1

comment : null
color : Red
root frequency : 9.703

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
cosy tocsy noe null null null null null null
9.703 9.703 9.703 9.703 9.703 9.703 9.703 9.703 9.703 9.703 1:VAL_*:HN
5.369 5.369 5.369 5.369 5.369 5.369 5.369 5.369 5.369 5.369 1:VAL_*:HA
1.786 1.786 1.786 1.786 1.786 1.786 1.786 1.786 1.786 1.786 1:VAL_*:HB
0.895 0.895 0.895 0.895 0.895 0.895 0.895 0.895 0.895 0.895 1:VAL_*:HG1*

The neighbor probabilities
--------------------------

Pattern Probability

The residue type probabilities
------------------------------

Residue Probability
LYS 1.000
VAL 1.000
ILE 1.000
THR 0.800
LEU 1.000

Since the frequencies were defined from the TOCSY experiment in the pattern, you need to edit the NOE and DQF frequencies. For this, use the Assign/Spin System/Tile+Show+Edit Frequencies menu item.

22.   Adjusting the spectrum-specific shifts for NOE

First you need to define the NOE spectrum-specific shifts for frequencies.

Select from the Experiments table the NOE spectrum and click the Draw icon.

Now select the Assign/Spin System/Tile+Show+Edit Frequencies menu item. Choose pa1 from the Pattern List and select Specific Shift and noe. Click OK.

You see the spectrum-specific shifts drawn on top of the tiled plot of the NOE spectrum, and an instruction message in the status bar (as well as in the title of the main window):

Click-drag-release to include the frequency you want to edit, or click <ESC> to quit.

Suppose the frequency 0.895 does not coincide with the NOE peaks well and you want to edit this frequency for the NOE spectrum. You may edit this frequency:

In any of the tiles that contain the 0.895 ppm frequency (e.g., the upper left-most tile), click-drag to draw a small rubber box that overlaps with the frequency 0.895 ppm. Release the mouse button. This selects the 0.895 ppm frequency for editing, and the cursor becomes a horizontal hair.

A new instruction message appears in the status bar (as well as in the title of the main window):

     Pick on the new position or hit <ESC> to quit.

Move the horizontal hair cursor to a location that aligns best with the NOE peaks. Click the left mouse button to select it.

The new shift is displayed, and a message appears with the new chemical shifts. You may have something similar to:

Selected frequency(s) to change:
D2: 0.892
Changed to new frequency(s):
D2: 0.9150143
Applied the changes to frequencies of experiment 'noe'.

You may repeat these steps for each frequency to obtain the best alignment for each frequency.

After you finish with the NOESY spectrum, you can repeat this for the DQF-COSY spectrum and check the results with the Assign/Report Spin System menu item.

You should notice that, under Frequencies, the specific shifts for NOE have been changed.

You now need to inspect the other prototype patterns and promote them to patterns, as was done in Steps 16, 17, and 18.

23.   Copying the 55th prototype pattern to the clipboard list

Select Assign/Frequency Clipboard/Zero Clipboard to clear the frequency clipboard. Next select Assign/Frequency Clipboard/Copy Proto to Clipboard and copy prototype pattern 55.

If TOCSY is not currently displayed, select it in the Experiment table and click the Draw icon. Next activate the spectral window and select the Assign/Frequency Clipboard/Strip Plot Clipboard menu item to display the strip plot.

You can see that, in the column of 9.194 ppm, there is an extra frequency around 2.5 ppm.

Use the Zoom icon in the main tool bar to zoom to the (9.19, 2.5 ppm) peak region.

Add this 2.5 ppm frequency to the frequency clipboard by selecting the Assign/Frequency Clipboard/Add One menu item, clicking the cursor on this peak, and then setting the Frequency parameter to W1 2.664909 and the Nucleus 1 to HX. Click OK.

Click <ESC> to terminate the frequency adding mode. Select Assign/Frequency Clipboard/Copy Clipboard to Pattern. Leave all the default settings in the dialog box and click OK. This promotes the frequencies in the clipboard to a new pattern, pa2.

Score the pattern as in Step 19. The result is:

Scoring pattern pa2
9.194 3.060 2.845 2.665
'H' 'H' 'H' 'H'
... vs cys | score=1 | aver.=1.168145 | matched atoms=4 / 4
... vs glu | score=0.6666667 | aver.=2.168036 | matched atoms=4 / 6
... vs lys | score=0.4444444 | aver.=1.916762 | matched atoms=4 / 9
... vs ser | score=1 | aver.=2.377159 | matched atoms=4 / 4
... vs tyr | score=0.3636364 | aver.=0.464548 | matched atoms=4 / 11
Scoring is done.
The most probable amino acid residue type is cysteine. Match this pattern against CYS:
Matching pattern pa2 versus cys
9.194 3.060 2.845 2.665
H H H H
HN 1.277 -7.486 -7.793 -8.050
HA 6.125 -2.053 -2.340 -2.580
HB1 16.668 0.526 -0.039 -0.513
HB2 15.879 -0.263 -0.829 -1.303

If you check the DQF-COSY spectrum (by selecting to display the COSY from the Experiment table, and then selecting pa2 in the Spinsystems table and clicking the Tile Plot icon), you can see that the frequency with the chemical shift of 2.845 has a cross peak with an amide proton (9.194 ppm), therefore this must be the alpha proton. HB2 is probably the frequency with 3.060 ppm, and HB1 the frequency with 2.665 ppm. Assign this pattern as described in Step 21. The result should be similar to:

LISTING FOR PATTERN pa2

comment : null
color : Red
root frequency : 9.194

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
cosy tocsy noe null null null null null null
9.194 9.194 9.194 9.194 9.194 9.194 9.194 9.194 9.194 9.194 1:CYSH_*:HN
3.060 3.060 3.060 3.060 3.060 3.060 3.060 3.060 3.060 3.060 1:CYSH_*:HB2
2.845 2.845 2.845 2.845 2.845 2.845 2.845 2.845 2.845 2.845 1:CYSH_*:HA
2.665 2.665 2.665 2.665 2.665 2.665 2.665 2.665 2.665 2.665 1:CYSH_*:HB1

The neighbor probabilities
--------------------------

Pattern Probability

The residue type probabilities
------------------------------

Residue Probability
LYS 0.444
CYS 1.000
TYR 0.364
GLU 0.667
SER 1.000

24.   Copying the 52nd prototype pattern to the clipboard list

Clear the clipboard using the Assign/Frequency Clipboard/Zero Clipboard menu item, then copy the 52nd prototype pattern to the clipboard with the Assign/Frequency Clipboard/Copy Proto to Clipboard menu item. Spawn a tile for the TOCSY spectrum as in Step 14, but use the clipboard as the source (Assign/Frequency Clipboard/Tile Clipboard).

You can see that, in the left-most tile in the row with 1.983 ppm, there is an extra frequency around 1.89 ppm.

Add this frequency to the clipboard list with the Assign/Frequency Clipboard/Add One menu item. Copy this clipboard list to the pattern pa3 and score it with Min atoms set to 6.

You should get the following output:

Scoring pattern pa3
8.928 3.972 1.983 1.498 1.674 1.893
'H' 'H' 'H' 'H' 'H' 'H'
... vs leu | score=1 | aver.=1.084693 | matched atoms=6 / 6
... vs lys | score=1 | aver.=0.6165203 | matched atoms=6 / 6
Scoring is done.

The most probable amino acid residue type is lysine. Select Assign/Residue Type/Match Residue Type to match against it, and then assign it.

Matching pattern pa3 versus lys
8.928 3.972 1.983 1.498 1.674 1.893
H H H H H H
HN 0.997 -6.628 -9.688 -10.434 -10.163 -9.826
HA 11.210 -0.590 -5.326 -6.481 -6.062 -5.540
HB1 18.942 5.900 0.666 -0.611 -0.147 0.429
HB2 20.935 6.359 0.509 -0.918 -0.400 0.244
HG1 19.277 6.569 1.469 0.226 0.677 1.238
HG2 20.265 6.870 1.495 0.184 0.659 1.251
HD1 30.242 9.592 1.304 -0.717 0.017 0.929
HD2 31.513 9.965 1.317 -0.791 -0.026 0.926
HE1 45.754 7.631 -7.669 -11.400 -10.046 -8.362
HE2 59.380 9.820 -10.070 -14.920 -13.160 -10.970
HZ* 1.398 -3.558 -5.547 -6.032 -5.856 -5.637

Further inspecting the TOCSY spectrum (strip plots), you can see two extra frequencies, at around 2.99 and 2.88 ppm, which you can add to the pattern with the Assign/Spin System/Add Frequency via Cursor menu item.

Now score this pattern again and store the result. Use 8 as the Min Atoms.

The result should be similar to:

Scoring pattern pa3
8.928 3.972 1.983 1.498 1.674 1.893 2.885 2.995
'H' 'H' 'H' 'H' 'H' 'H' 'H' 'H'
... vs lys | score=0.8 | aver.=0.6080632 | matched atoms=8 / 10
Scoring is done.

which clearly shows that the original assumption - that the pattern is a listen type - was a valid one (leucine is now ruled out, although it was possible from the score itself).

Unambiguous assignment is possible only for the amide and alpha proton, therefore the pattern listing will show the following results:

LISTING FOR PATTERN pa3

comment : null
color : Red
root frequency : 8.928

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
cosy tocsy noe null null null null null null
8.928 8.928 8.928 8.928 8.928 8.928 8.928 8.928 8.928 8.928 1:LYS+_*:HN
3.972 3.972 3.972 3.972 3.972 3.972 3.972 3.972 3.972 3.972 1:LYS+_*:HA
1.983 1.983 1.983 1.983 1.983 1.983 1.983 1.983 1.983 1.983 1:LYS+_*:HX
1.498 1.498 1.498 1.498 1.498 1.498 1.498 1.498 1.498 1.498 1:LYS+_*:HX
1.674 1.674 1.674 1.674 1.674 1.674 1.674 1.674 1.674 1.674 1:LYS+_*:HX
1.893 1.893 1.893 1.893 1.893 1.893 1.893 1.893 1.893 1.893 1:LYS+_*:HX
2.995 2.995 2.995 2.995 2.995 2.995 2.995 2.995 2.995 2.995 1:LYS+_*:HX
2.885 2.885 2.885 2.885 2.885 2.885 2.885 2.885 2.885 2.885 1:LYS+_*:HX

The neighbor probabilities
--------------------------

Pattern Probability

The residue type probabilities
------------------------------

Residue Probability
LYS 0.800

25.   Copying the 49th prototype pattern to the frequency clipboard

Follow the procedure described in Step 25 to copy prototype pattern 49 to the clipboard. Then spawn a tile plot from the clipboard for the TOCSY spectrum using the Assign/Frequency Clipboard/Tile Clipboard menu item. You can see that a frequency at 0.853 ppm was missed during the automated routine. Add it to the clipboard and then copy the list to the pa4 pattern. Set the spectrum-specific shifts. Now score and store the result of the pattern using 5 as the Min Atoms:

Scoring pattern pa4
9.094 4.049 2.545 1.366 0.846 0.749
'H' 'H' 'H' 'H' 'H' 'H'
... vs ile | score=0.7142857 | aver.=1.097882 | matched atoms=5 / 7
... vs leu | score=1 | aver.=1.303445 | matched atoms=6 / 6
... vs lys | score=1 | aver.=1.344301 | matched atoms=6 / 6
... vs pro | score=0.7142857 | aver.=1.726693 | matched atoms=5 / 7
... vs val | score=0.8333333 | aver.=0.9049344 | matched atoms=5 / 5
Scoring is done.

Since there are clearly at least six frequencies in the pattern, the valine possibility can be dropped. Also, since there is an HN frequency, the proline can be excluded. The remaining possibilities are leucine, lysine, and isoleucine. Since the frequencies with 0.836 and 0.735 ppm are methyl groups, the lysine can also be excluded.

From the Experiments table, select the DQF spectrum. Click the Draw icon to display the COSY spectrum.

From this you can see that the frequency with 2.545 ppm belongs to a possible beta methine or methylene proton. This is connected with a strong COSY interaction with the methyl frequency at 0.846 ppm, which is only possible in an isoleucine spin system. Therefore, this spin system is an isoleucine type.

In the Spinsystems table, select the Spinsystem/List Residue Type menu item.

Select the Assign/Residue Type/Set Residue Type menu item for pa4 and choose Assign One as the Action. Choose ILE from the list and click OK.

A message appears:

     Residue type of pattern pa4 is set to ile

You can verify the results with the Assign/Residue Type/Show Residue Type menu item.

The output is:

     The probability for pa4 to be ILE is : 1.000

After matching the pattern against the ILE residue, you can assign the frequencies.

Matching pattern pa4 versus ile
9.094 4.049 2.545 1.366 0.846 0.749
H H H H H H
HN 1.283 -5.724 -7.812 -9.450 -10.163 -10.307
HA 9.431 -0.271 -3.163 -5.431 -6.417 -6.617
HB 19.795 6.159 2.095 -1.092 -2.478 -2.759
HG11 30.977 11.573 5.788 1.254 -0.719 -1.119
HG12 23.981 8.216 3.516 -0.169 -1.772 -2.097
HG2* 34.600 13.579 7.312 2.400 0.262 -0.171
HD1* 33.416 13.236 7.220 2.504 0.452 0.036

Since you saw from the DQF spectrum that the frequency with 2.545 ppm is the beta methine proton and that it has a cross peak with the methyl at 0.846 ppm, the assignments are as follows:

LISTING FOR PATTERN pa4

comment : null
color : Red
root frequency : 9.094

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
cosy tocsy noe null null null null null null
9.094 9.094 9.094 9.094 9.094 9.094 9.094 9.094 9.094 9.094 1:ILE_*:HN
4.049 4.049 4.049 4.049 4.049 4.049 4.049 4.049 4.049 4.049 1:ILE_*:HA
2.545 2.545 2.545 2.545 2.545 2.545 2.545 2.545 2.545 2.545 1:ILE_*:HB
1.366 1.366 1.366 1.366 1.366 1.366 1.366 1.366 1.366 1.366 1:ILE_*:HG12
0.84653 0.853 0.853 0.853 0.853 0.853 0.853 0.853 0.853 0.853 1:ILE_*:HG2* 0.749 0.749 0.749 0.749 0.749 0.749 0.749 0.749 0.749 0.749 1:ILE_*:HD1*

The neighbor probabilities
--------------------------

Pattern Probability

The residue type probabilities
------------------------------

Residue Probability
ILE 1.000

26.   Copying the 4th prototype pattern to clipboard list

This time you will promote (copy) a prototype pattern directly to a pattern (spin system).

Select the 4th prototype pattern in the Protopatterns table by clicking the fourth row.

Go to the table menubar and select the ProtoPattern/Promote One Proto To Spin System item.

In the control panel,, leave all the default settings unchanged. Click OK.

This adds a new spin system, pa5, in the Spinsystems table.

Select Assign/Residue Type/Score Residue Type to score and store the result of pattern pa5. Use 5 as the Min Atoms:.

The residue type scoring appears in the output window as:

Scoring pattern pa5
9.274 5.022 3.288 2.526
'H' 'H' 'H' 'H'
... vs asn | score=1 | aver.=1.229159 | matched atoms=4 / 4
... vs asp | score=1 | aver.=1.165284 | matched atoms=4 / 4
... vs cys | score=1 | aver.=0.7924712 | matched atoms=4 / 4
... vs lys | score=0.4444444 | aver.=1.975681 | matched atoms=4 / 9
... vs phe | score=1 | aver.=0.78622 | matched atoms=4 / 4
... vs ser | score=0.8 | aver.=1.212075 | matched atoms=4 / 5
... vs thr | score=0.8 | aver.=1.514605 | matched atoms=4 / 5
... vs tyr | score=1 | aver.=1.10682 | matched atoms=4 / 4
Scoring is done.

with the most likely candidates as phenylalanine and cysteine. Match against these two residues:

Matching pattern pa5 versus phe
9.274 5.022 3.288 2.526
H H H H
HN 1.018 -4.298 -6.465 -7.418
HA 9.550 0.692 -2.921 -4.508
HB1 23.050 7.864 1.671 -1.050
HB2 21.764 6.579 0.386 -2.336
HD1 7.867 -7.881 -14.304 -17.126
HE1 6.813 -7.360 -13.140 -15.680
HZ 7.117 -7.545 -13.524 -16.152
HE2 6.813 -7.360 -13.140 -15.680
HD2 7.867 -7.881 -14.304 -17.126
HD* 7.867 -7.881 -14.304 -17.126
HE* 6.813 -7.360 -13.140 -15.680

Matching pattern pa5 versus cys
9.274 5.022 3.288 2.526
H H H H
HN 1.391 -4.683 -7.160 -8.249
HA 6.232 0.563 -1.749 -2.765
HB1 16.879 5.689 1.126 -0.879
HB2 16.089 4.900 0.337 -1.668

Since we do not know at this stage what the residue type is, we can leave that undetermined and let the automated routines come up with a possible answer later.

27.   Finding the sequential connectivities

Once a set of patterns is determined, the next step is to connect these patterns. This is possible with the neighbor-finding algorithm. Generally, it is very important that your spectrum-specific shifts for the NOE spectrum be set for all patterns, as well as for the root frequencies, before you attempt to perform this action.. In this tutorial, however, proceed without doing that for each pattern. Also, make sure to select the NOE spectrum from the Experiments table.

If NOESY is not displayed, select the NOE spectrum from the Experiments table and click the Draw icon.

Select the Assign/Neighbor/Find Neighbor Via 2D NOE menu item. Set these values:

Experiment noe
Root Frequency Tolerance 0.015
Frequency Collapse Tolerance 0.015
Output Level
Low

Leave the other parameters at their default values and click More... to see the default parameters.

In the next control panel, leave the defaults as they are and click OK.

After one or two seconds the results are printed and stored:

Spectrum :(zn.mat)
Tolerances :( 0.010 0.010 )
Root & Collapse tolerances: 0.0100000 0.0150000
Pattern :(ALL)
Now at pattern :( 1 )
Use :( 5.369 1.786 0.885 )
Neighbors :( 2 )
Scores :( 1.000 )
Now at pattern :( 2 )
Use :( 3.060 2.845 2.665 )
Neighbors :( 3 )
Scores :( 1.000 )
Now at pattern :( 3 )
Use :( 3.972 1.983 1.498 1.674 )
Neighbors :( 4 )
Scores :( 1.000 )
Now at pattern :( 4 )
Use :( 4.049 2.545 1.366 0.749 )
Neighbors :( 5 3 )
Scores :( 0.600 0.400 )
Now at pattern :( 5 )
Use :( 5.022 3.288 2.526 )
No candidate neighbors

From this listing you can see which pattern is neighbor to which, i.e., what the sequential connection is (i - i+1). For example, pa2 is neighbor to pa1, pa3 is neighbor to pa2, pa4 is neighbor to pa3, and pa5 is neighbor to pa4.

You can also check the stored values by selecting the Assign/Neighbor/List Neighbor menu item (or by using the Spinsystem table's Spinsystem/List i+1 Neighbors control, having selected the first pattern from the table).

Click pa1 and leave i - i+1 for Order.

Click OK.

In the output window you see:

     The possible neighbors for pattern pa1 are: 
pattern pa2 with probability: 1.0000

28.   Visually verifying the results of neighbor detection

First click the third row (pa3) in the Spinsystems table, then <Ctrl>-click to select the fourth row (pa4).

Click the Tile Plot icon.

You now should see the results as a tile plot of the inter-pattern peaks.

Select Contour from the popup in the main icon bar to go to the contour plot.

Go to the Spinsystems table and click the Draw icon to overlay frequencies on the plot.

Now you see the frequencies displayed on top of the tile plot.

Inspecting the plot reveals that there are really inter-residue (inter-pattern) cross peaks. There is a well-defined cross peak at frequencies 8.928 and 9.094 ppm, which is an amide-amide cross peak between the two neighboring residues (dHN(LYS)HN(ILE)). Also, there is a cross peak between 3.972 and 9.094 ppm, which is an alpha-amide cross peak (dHa(LYS)HN(ILE)). There is a cross peak at 1.893 and 9.094 ppm, which can be a beta-amide cross peak, since from residue matching you can see that this frequency likely belongs to a beta proton in the lysine residue. These two (three) interactions usually determine a sequential connectivity.

After neighbor detection, the next step is to match the found patterns against the known amino acid sequence.

29.   Matching the found patterns against the known amino acid sequence

Select the Assign/Sequential/Systematic Search menu item. Leave the settings in the control panel at their defaults, except for the following:

Last residue (#) to consider 53
Max # of assignments to generate 100
Output level Medium

Click OK.

After a few seconds, the suggestion is ready. The output contains information about several steps in the automated routine:

     Constructing assignment-probability matrix
Probs for ALA :( 0.000 0.000 0.000 0.000 0.000 )
Probs for LYS :( 0.990 0.000 0.800 0.000 0.000 )
Probs for TRP :( 0.000 0.000 0.000 0.000 0.000 )
Probs for VAL :( 0.990 0.000 0.000 0.000 0.000 )
Probs for CYS :( 0.000 0.990 0.000 0.000 0.990 )
Probs for LYS :( 0.990 0.000 0.800 0.000 0.000 )
Probs for ILE :( 0.990 0.000 0.000 0.990 0.000 )
Probs for CYS :( 0.000 0.990 0.000 0.000 0.990 )
Probs for GLY :( 0.000 0.000 0.000 0.000 0.000 )
...

Constructing neighbour-probability matrix
Nbrs for null :( 0.000 1.000 0.000 0.000 0.000 )
Nbrs for null :( 0.000 0.000 1.000 0.000 0.000 )
Nbrs for null :( 0.000 0.000 0.000 1.000 0.000 )
Nbrs for null :( 0.000 0.000 0.400 0.000 0.600 )
Nbrs for null :( 0.000 0.000 0.000 0.000 0.000 )

Generating the assignments ...
... found 0 stretches starting at residue 1
... found 0 stretches starting at residue 2
... found 0 stretches starting at residue 3
... found 1 stretches starting at residue 4
... found 1 stretches starting at residue 5
... found 0 stretches starting at residue 6
...
Number of assignments generated :( 2)
Buffer usage pointers (%) :( 0.040 )
Buffer usage assignments (%) :( 0.002 )
Sorting out the generated assignments
Assignments left :( 1 )
assignment # 1 -- length = 5 residues
...stretch of residues = 4 - 8 total scores:4.76 3.60
Residues:VAL_4 CYSH_5 LYS+_6 ILE_7 CYSH_8
Patterns: 1 2 3 4 5
Scores: 0.99 0.99 0.80 0.99 0.99
I>I+1: 1.00 1.00 1.00 0.60
The Pattern Suggest Assignment took 1 seconds!

The program thus suggests that pa1 belongs to residue 4 (VAL_4), pa2 belongs to residue 5 (CYSH_5), pa3 belongs to LYS+_6, pa4 belongs to ILE_7, and pa5 belongs to CYSH_8. The residue type of this latter spin system was in question - based on frequencies, the program could not distinguish between cysteine and phenylalanine. Now this ambiguity is resolved through the use of systematic search.

A new spreadsheet came up - one which contains this possible sequential assignment in tabular form: Stretches. Now you can make sequence-specific assignments for the known frequencies.

30.   Making the sequence-specific assignment for pa1

Now you have two options:

a. Use the Stretches table to make a quick assignment, then recheck the results and possibly edit the assignments using the Spinsystems table.

b. Use the following sequence of commands:

Select the Assign/Assign Spin System/Frequency menu item. Select pa1 from the list of patterns and click Next.

Following the procedures in Step 21, you next assign the sequence-specific assignment for each frequency of pal.

Click 9.703 ppm in Frequency list and then click Select.

Now select VAL from the Residues list. Click Filter.

Next select 4 from the #'s list, and HN from the Protons list. Click Build.

Select 1:VAL_4:HN from the Possible Assignment list.

Click Add. Now select the next frequency at 5.369 ppm, and use the same procedure to reassign this to 1:VAL_4:HA.

Alternatively, you can select any item in the Assignments list and then edit it in the Atom Spec box. Next, click ADD to accept it.

You can do the assignments on the Spinsystems table, too. For this, you just edit the fields next to the resonances.

After finishing the last frequency, click New Pattern and make sequence-specific assignments for each pattern.

Once you assign the frequencies, you must transfer these assignments to peaks in order to use them together with volume measurements of those peaks in a refinement procedure.

31.   Checking the NOE peaks

Make sure that the NOE spectrum is active. Display the full spectrum by pressing <Ctrl>-f while in intensity mode.

Displaying a full-spectrum contour plot with several contour levels can be time consuming. Using the hot keys <Ctrl>-i is an alternative.

Select the Preference/Peak Display menu item to see whether any peaks are assigned.

In the first control panel, set the Coloring Mode parameter to By Assignment and click Draw.

In the following control panel, set these values:

Fully assigned Green
Partially (D1) assigned Magenta
Partially (D2) assigned Cyan
Multiply assigned Blue
Not assigned Red

Click OK.

All the peaks should now be red, indicating that none of them are assigned yet, although some of the frequencies are assigned.

Now you need to transfer frequency assignments to peak assignments.

32.   Automatically generating peak assignments from frequency assignments

Select the Assign/Peak Assign/Autoassign Peaks menu item and set these values:

Rejection Cutoff 8
Output Level Quiet
Unambiguous Cutoff 6.0
Strictly Enforce Distance Yes
Peak Entity xpk:noe.
Multiple Assign off
Skip Fully Assigned off
Skip Multiple Assigned off

Click OK.

In a few seconds you should see output similar to the following:

     Assign peaks for spectrum :(noe)
Tolerances :( 0.010 0.010 )
Spins (h h)
Folding (0 0 )
Transfers ( N )
Nr of peaks unambiguously assigned :( 95 )
Nr of peaks with competing assmnts :( 0 )
Nr with no or too many assignments :( 1787 )
The peak auto assignment took 9 seconds

The cross peaks have different colors, depending on the assignment status: green for fully assigned peaks, red for non-assigned peaks, blue for multiply assigned peaks, and turquoise and purple for partially assigned peaks. You should see several green peaks, with the majority of peaks still being red.

Next you go back and check whether the peaks belonging to different patterns were assigned correctly.

33.   Checking the peak assignment for pattern 1

Go to the Spinsystems table and select the first pattern (pa1). Then click the Tile Plot icon.

Now draw the peaks if they are not drawn, using the View/Draw Peaks menu item. Notice that the different peaks are in different colors, depending on assignment status.

The peak at 9.698 and 5.370 ppm is now green, showing that the peak was assigned along both frequencies. The peak at 5.37 and 9.64 ppm and the symmetric peak at 9.64 and 5.37 ppm are both red, showing that the peaks have not been assigned yet.

34.   Checking the inter-residue peak assignment

Next you follow a similar procedure for inter-residue peaks.

Go to the Spinsystems table and select the first pattern by clicking its row. Then <Ctrl>-click to select the second pattern (pa2).

Click the Tile Plot icon.

Press <Ctrl>-k if peaks are not drawn.

The peak at 5.369 and 9.194 ppm is green, denoting that this is a fully assigned inter-residue peak between VAL_4 and CYSH_5.

Now you can list the peak.

Select the Assign/List Peak menu item and, with the resulting cross-hair cursor, pick the peak at 5.369 and 9.189 ppm.

Click the <Esc> key to exit the peak listing mode.

You now should see output in the output window that looks like:

Peak # 133 Intensity = 0.336250e+06
Dimension Position (ppm) Width (Hz) Peak Assignment
W2 5.36633 20.95018 1:VAL_4:HA
W1 9.18967 17.5624 1:CYSH_5:HN
Dimension Frequency Assignment Distance(ppm) Pattern id
W2 1:VAL_4:HA 0.003056 pa1
W1 1:CYSH_5:HN 00.0065804 pa2

This indicates an daN(i,i+1) NOE connectivity. If you have the corresponding peak table open as a spreadsheet (Peaks-xpk:noe), this peak is highlighted in the table.

Note the red peak at around 9.7 and 9.1 ppm, which is in the lower-left box of the tile display - if you list it with the Assign/List Peak menu item, you see that the two frequencies defining this peak are assigned to 1:VAL_4:HN and 1:ILE_7:HN but that the peak itself was not assigned:

Peak # 154 Intensity = 0.464310e+05
Dimension     Position (ppm)      Width (Hz)   Peak Assignment
W2 9.70856 21.65628
W1 9.08714 20.65723

Dimension  Frequency Assignment  Distance(ppm)  Pattern id
W2           1:VAL_4:HN 0.0030603 pa1
W1           1:ILE_7:HN 0.0090561 pa4

This is because the two atoms are farther apart than the NOE cutoff used in automated assignment (8 ). You can check this with the following action.

Select the Measure/Distance/Separation menu item or click the Measure Distance icon. Click this peak.

You see the following output in the output window:

     Peak # 154

Frequency Assignment:
W2 W1 Distance (A)
1:VAL_4:HN 1:ILE_7:HN 11.1772

This proves that the peaks were not assigned because the distance criterion was not met.

Turn off the tile mode using the View/Tile Plot/Tile Plot menu item.

To generate structures you need to assign all the peaks.Usually the peak assignment should be done on an NOE spectrum where buildup (i.e., multiple mixing time experiments) information is also available. There is a spectrum - a 450-ms mixing-time NOESY experiment which is defined in the following database.

35.   Reading in the database containing fully-assigned patterns

Select the File/Open menu item. Select DBA for the File Type and select the zn_model.dba file from the list. Click OK.

If it prompts you to save changes to the previous DBA file you opened, click Save Changes to save them.

All tables are automatically closed. Select Assign/Experiment to open the Experiments table. Click OK to the next dialog box. Select Edit/Spin Systems to open the Spinsystems table.

Now select the buildup experiment using the Experiments table, highlighting the fourth row and clicking the Draw icon. Click the Full Plot icon to draw the full plot if necessary.

The spectrum-specific shifts for this experiment are not exactly set yet, you need to adjust them in the next step:

Select the Assign/Spin System/Auto Update Specific Shifts menu item. Check Use All Patterns. Set the Spectrum Specific Shift parameter to the buildup experiment. The D1 and D2 Tolerance should be set to 0.015. Click OK.

The next step is to assign all the peaks with the help of these newly defined spectrum-specific chemical shifts.

36.   Assigning the buildup automatically

Select the Assign/Peak Assign/Autoassign Peaks menu item and be sure that these values are set:

Rejection Cutoff 8.0
Unambiguous Cutoff 6.0
Strictly Enforce Dist on
Peak Entity xpk:buildup
Multiple Assign off
Skip Fully Assigned off
Skip Multiply Assigned off
Output Level Quiet

Click OK.

After one or two minutes the auto-assignment is done. You should see something like this in the output window:

     Generating automatic assignments
Please wait ...
Press <Esc> to quit.
Assign peaks for spectrum :(buildup)
W1 W2
Spins :(hh )
Folding :( 0 0 )
Transfers :(N )
Tolerances :( 0.010 0.010 )
Nr of peaks unambiguously assigned :( 858)
Nr of peaks with competing assmnts :( 0 )
Nr of peaks with no new assignment :( 1104 )

The peak auto assignment took 81 seconds

The following step is to define NOE distance restraints from this spectrum. In restraint definition, the first step is to define a scalar peak, for which the distance between the atoms it is assigned to is fixed. There are several ways of doing this, but for now we will demonstrate with a fixed HB1-HB2 peak.

37.   Defining the scalar peak

Select the Peaks/Find menu item. Set these values:

Find Peak By Number
Action Zoom+Color
Peak ID 1153

Click OK.

List this peak with the Assign/List Peak menu item:

Peak # 1153 Intensity = 0.2238639e+07
Dimension Position (ppm) Width (Hz) Peak Assignment
F2 2.255588 32.96875 1:ASP-_13:HB1
F1 2.859035 36.89453 1:ASP-_13:HB2
Dimension Frequency Assignment Distance(ppm) Pattern id
F1 1:ASP-_13:HB2 0.001035 pa6
F2 1:ASP-_13:HB1 0.4119873e-03       pa6
F1 1:ASP-_35:HB2 0.96488e-03        pa12

This will be the scalar peak.

Select the Measure/Scalar_Normalize menu item. In the control panel, set the Add One option and click OK.

In the next control panel, set the values:

Peak Name D1 1:ASP-_13:HB1
Peak Name D2 1:ASP-_13:HB2
Distance 1.75

Click OK.

Now set the intensity plot (if you were in contour plot) and draw a full plot (View/Limits/Full Limits).

38.   Defining the restraints

Select the Measure/DISCOVER Restraints menu item. In the first control panel, set Restraint Class to NOE Distance and Action to Define, then click OK.

Set these values in the next control panel:

Action New
Scalars etc:scalars
Lower Force Const 30.0
Upper Force Const 30.0
Maximum Force 1000.0
Calculation Method Single tm
Mixing Time 0.1
Symmetry Selection Use Weaker
Method S-M-W Bins
Partial Overlap Use as Qual
Are Threshold 50

Click OK.

In the third control panel, set these values:

Strong -1.0 2.5
Medium -1.0 3.5
Weak -1.0 6.0

Click OK.

In the fourth control panel, leave Lower Bound at -1.0 and Upper Bound at 6.0 for the overlapped peaks. Click OK.

Yellow footprints appear on the plot, indicating the peaks from which restraints are generated. At the end of the procedure a message appears:

474 NOE distance restraints calculated
From these 51 were qualitative distance restraints because of partial overlap
0 peaks were discarded because of assignment problems

After you have finished peak assignment and restraint generation, you can move on to generate structures in NMR Refine. Therefore, the last step is to write out a database that you can import to Insight II.

39.   Writing the database

Select the File/Export/Restraints menu item. Set Peaks and Resonances and Restraints to on.

For all the filenames (Peak Intensity File, Chemical Shift File, Assignment File, Restraint File) enter: znrdlec and set the Type to DISCOVER.

Click OK.

This action writes out the znrdlec.pks, znrdlec.ppm, znrdlec.asn, and znrdlec.rstrnt files.

After running DGII or simulated annealing, the first structures are generated. A new set of assignments can be generated based on this new structure(s). First you can redefine the molecular structure and then rerun auto-assignment.

40.   Redefining the coordinates

Select the Assign/Read Coordinates menu item. From the list select znrddg.car and click OK.

This replaces the linear-chain coordinates of Zn-rubredoxin with the first DG-II structure coordinates.

41.   Rerunning autoassignment for the buildup

First unassign the peaks by selecting Assign/Peak Assign/Unassign Peaks and leave the peak entity as xpk:buildup. Click OK.

Select the Assign/Peak Assign/Autoassign Peaks menu item and set these values:

Rejection Cutoff 8.0
Strictly Enforce Dist on
Unambiguous cutoff 6.0
Peak Entity xpk:buildup
Multiple Assign off
Skip Fully Assigned off
Skip Multiply Assigned off
Output Level Quiet

Click OK.

In a few minutes you see:

     Generating automatic assignments
Please wait ...
Press <Esc> to quit.
Assign peaks for spectrum :(buildup)
W1 W2
Spins :(hh )
Folding :( 0 0 )
Transfers :(N )
Tolerances :( 0.010 0.010 )
Nr of peaks unambiguously assigned :( 1123 )
Nr of peaks with competing assmnts :( 0 )
Nr of peaks with no new assignment :(      839 )

The peak auto assignment took 87 seconds

More than 250 new peak assignments were made based on the preliminary DG-II structure.

42.   Regenerating the restraints

Select the Measure/DISCOVER Restraints menu item. In the first control panel, set Restraint Class to NOE Distance and Action to Define. Click OK.

Set these values in the second control panel:

Action New
Scalars etc:scalars
Lower Force Const 30.0
Upper Force Const 30.0
Maximum Force 1000.0
Calculation Method Single tm
Mixing Time 0.1
Symmetry Selection Use Weaker
Method S-M-W Bins
Partial Overlap Use as Qual
Are Threshold 50

Click OK.

In the third control panel, set these values:

Strong -1.0 2.5
Medium -1.0 3.5
Weak -1.0 6.0

Click OK

And in the fourth control panel, leave Lower Bound at -1.0 and Upper Bound at 6.0 for the overlapped peaks. Click OK.

Yellow footprints again appear on the plot, indicating the peaks from which restraints are generated. At the end of the procedure a message appears:

633 NOE distance restraints calculated
From these 61 were qualitiative distance restraints because of partial overlap
0 peaks were discarded because of assignment problems

43.   Redefining restraints

Typically, after a DGII or simulated annealing run you need to analyze your restraints. This can be done in Insight II, and the results can be printed as a file containing a list of restraints that are violated in multiple structures. In FELIX you can then use that file to help you to redefine or reassign erroneous assignments or restraints. This is what you do in the next step.

Select the Measure/DISCOVER Restraints menu item again. In the control panel set Restraint Class to NOE Distance and Action to Redefine.

Click OK.

In the second control panel, enter zn_viol01.txt as the Filename as and leave the other parameters at their defaults. Click OK.

The program now brings up a new spreadsheet containing the distance restraint violations - Violations. In this table you can zoom in on the peak defining the first problematic restraint and can also see the values of the restraint and the violations.

Select the first row in the Violations table and click the Zoom icon. The restraint for 1:GLU-_47HN and 1:GLU-_47:HG2 which had a restraint between 1.8 and 4.5 was violated in 14 conformations out of a total of 20, and the violation average was 0.17 . The average distance measured in the 20 conformation is 4.64 and the calculated distance based on ISPA is 2.78 . You can see that this peak is heavily overlapped, and the symmetric peak has not been assigned at all (Click the Symmetric Peak icon to check). Since this restraint is very unreliable, you may want to delete it.

Go to the Violations table and select the Violation/Delete Restraint menu item.

The output window now says:

     Item 320 deleted from biosym:noe_dist.

Now you can see in the NOE-Restraints table that this restraint was indeed deleted from the database.

Now select the second violation from the Violations table and click the Zoom icon.

A new peak appears in the spectrum, which is the next problematic restraint. This is a well-defined peak and the distance calculated on the symmetric peak is larger than the one from this peak (use Violation/ Calculate Distance in the Violations table) and also larger than the original restraint was:

Peak 1043 1:GLY_42:HN - 1:CYSH_38:HB2 distance: 3.202873
Symmetric peak 369 distance: 3.564213

You may want to simply increase the bounds for this as in the following step.

Select the Violation/Redefine Bounds menu item from the Violations table. Set Lower Bound to -1.0 and Upper Bound and Upper Bound with Correction to 5.0. Click OK.

The program now informs you:

     Item 243 updated in biosym:noe_dist.

Since there is no other violated restraint left in this file, this finishes the redefinition.

44.   Calculating the chemical shift index

The chemical shifts of certain spins can be informative about regular secondary structural elements. This can be exploited as shown in the following step.

Since this is homonuclear data, select the Assign/Chemical Shift Index/HA Chemical Shift Index menu item. Set these values:

Library Name csi.rdb
CSI File zn:hacsi
File to Export csi.txt
Molecule Name ZNRDDG

Click OK.

In few moments the calculation is done and a spreadsheet appears (HA-CSI) showing the residues, the assigned HA chemical shifts, and the CSI index and grouping, as well as the Richardson classification. By browsing through the table you can see regions that were found to be beta-sheets or alpha-helices. The program also wrote a file csi.txt with this classification to the disk. This file can be imported into Insight II and can (for example) be rendered on the ZNRDDG molecule.

45.   Writing the database

Select the File/Export/Restraints menu item. Set Peaks and Resonances and Restraints to on.

For all the filenames (Peak Intensity File, Chemical Shift File, Assignment File, Restraint File) enter: znrddg, set the Type to DISCOVER, and click OK.

This action writes out the znrddg.pks, znrddg.ppm, znrddg.asn, and znrddg.rstrnt files.

46.   Exiting FELIX

To exit FELIX, select the File/Exit menu item.


Lesson 2: Heteronuclear double-resonance 3D assignment strategy

This tutorial shows typical steps involved in assignment of a singly- labelled protein. The data set is the 15N-HSQC, 15N-HMQC-TOCSY, and 15N-HMQC-NOE spectra of the 15N-enriched MCP-1 protein from P. J. Domaille (DuPont Merck, Wilmington) and T. Handel (University of California, Berkeley).

The topics covered in this lesson are:

1.   Setting up for the lesson

If not done yet, set up the tutorial files as described in "Setting up tutorial files" in the preface, How to use this book.

The files for this lesson are all located in the Assignment\Lesson2 folder

2.   Starting FELIX

Start FELIX by double-clicking the FELIX icon on your desktop, or by clicking the Start button on the Windows taskbar, then selecting Programs/Accelrys FELIX 2004/FELIX 2004. If FELIX prompts you to restore from last session, click Cancel.

Change your Current Working Directory to C:\Felix_Practice\Assign\Lesson2\ using the Preference/Directory... command. Build a new database by opening the File/New... command and choosing Create a new matrix or DBA file. Make sure the File Type is set to DBA(*.dba). Enter mcp.dba and click OK.

3.   Setting up the database

Select the Assign/Project menu item.

FELIX informs you that no project was found in the database.

Click OK to acknowledge that you want to build a new project.

FELIX then asks you for a new project name.

The default name in this new dialog box is asg:project. Enter a different name if you want (for example, mcp:project)

.

In the second control panel, set this value:

File name mcp1_lec.car
File type Insight Molecule (*.car)

Click OK to build the entities and read in the molecule.

After this step is successfully completed, a library should be defined. The library is an ASCII file, as described in the Assign/Define Library section of the separate FELIX User Guide. FELIX contains a standard library (pd.rdb) which you should read in.

In the third control panel, toggle the Define Library From File parameter on and click OK.

In the fourth control panel, select pd.rdb and click OK.

4.   Adding experiments to the projects

Select the Assign/Experiment menu item to define new experiments in the assignment database. When the list of names of matrices appears, select hsqc.mat (the 15N-HSQC spectrum).

Click OK.

Set these values for the plot, in the 2D Display Parameters control panel:

Contour Threshold 0.02
Color Scheme Blue/Green
Axis type D1 ppm
Axis type D2 ppm
D1 Scale 1
D2 Scale 2

Leave the other parameters at their default values and select Apply.

Click OK when the message box appears.

If you want, you can change the display parameters by going to the Experiments table and using its Experiment/Change Attribute menu item later.

The program plots a density or contour plot of the 15N-HSQC spectrum using the parameters you defined.

Now another control panel appears.

Set the parameters to these values:

Title hsqc

Use Default Names on
Type 2D N-15 HSQC
Temperature 298
pH 7
Solvent Water
D1 Nucleus Proton
D2 Nucleus Nitrogen
D1 Tol 0.02
D2 Tol 0.07
D1 Fold No
D2 Fold No
W1 D2
W2 D1
W1-W2 Transfer J-coupled
# of J Steps 1

Click OK.

The spectrum-specific tolerances are important to define and are used in many automated and semi-automated procedures.

A new window containing an Experiments table is open and displayed to the left of the spectral window. Note that, by default, whenever a new window (table or spectral) is open, FELIX automatically re-arranges the layout of the windows. You can turn off this feature by selecting Preference/Frame Layout from the main menu and set Action to None. You can also do the automatic re-arrangement at anytime by selecting Window/Auto Arrange.

Note: When one or more table windows are open, only the menu and tool bar of the currently activated window are visible. If you want to select a certain menu item or tool bar icon, be sure to click the corresponding window first to activate its menu and tool bar (if any).

5.   Adding the 15N-HMQC-TOCSY experiment to the projects

Activate the Experiments table so that its own menu is displayed. Select the Experiment/Add menu item to define the next experiment in the assignment database. When the list of names of matrices appears, select mcpn15tocsy.mat (the 15N-HMQC-TOCSY spectrum).

Click OK.

Set these values for the plot using the 3D Display Parameters control panel:

Contour Threshold 0.5e-5
Color Scheme Magenta
Number Of levels 16
Negative Levels Off
Axis type D1 ppm
Axis type D2 ppm
Axis type D3 ppm
D1 Scale 1
D2 Scale 1
D3 Scale 1

Leave the other parameters at their default values and select Apply.

Click OK when the message box appears.

If you want, you can change the display parameters using the Experiment/Change Attribute menu item in the Experiments table later.

The program plots a density plot or contour plot of the first D1-D2 plane of the 15N-HMQC-TOCSY spectrum using the parameters you defined.

Now another control panel appears.

Set the parameters to these values:

Title tocsy

Use Default Names on
Type 3D HSQC-TOCSY
Temperature 298
pH 7
Solvent Water
D1 Nucleus Proton
D2 Nucleus Proton
D3 Nucleus Nitrogen
D1 Tol 0.02
D2 Tol 0.04
D3 Tol 0.1
D1 Fold No
D2 Fold No
D3 Fold No
W1 D3
W2 D1
W3 D2
W1-W2 Transfer J-coupled
# of J Steps 1
W2-W3 Transfer J-coupled
# of J Steps 7

Click OK.

6.   Repeating Step 4 for the 15N-HMQC-NOE spectrum

Again activate the Experiments Table and select the Experiment/Add menu item.

Set these values:

15N-HMQC-NOE mcpn15noe.mat
plotting parameters:
Contour Threshold 0.2e-4
Number Of Levels 16
Level Multiplier default setting
Color Scheme Cyan
Axis Type ppm
Negative Levels Off Off
D1 Scale 1.0
D2 Scale 1.0
D3 Scale 1.0

Click OK.

In the next control panel select these values:

Experiment Title noe
Use Default Names on
Type 3D HSQC-NOESY Temperature 298
pH 7
Solvent Water
D1 Nucleus Proton
D2 Nucleus Proton
D3 Nucleus Nitrogen
W1 D3
W2 D1
W3 D2
W1-W2 Transfer J-coupled
W2-W3 Transfer NOE
# of J Steps 1
Mixing time 0.1
spectrum specific tolerances
D1 0.02
D2 0.025
D3 0.1

Click OK.

7.   Reading in the peaks

Select the first row in the Experiment table by clicking it to chose the hsqc experiment. Then click the Draw icon.

Activate the spectral window so that the main menu is displayed. Select the File/Import/Peaks menu item. Set the Selection parameter to hsqc.xpk. Leave the FELIX Peak Table Name parameter at its current value (xpk:hsqc) and the Peak File Type as FELIX Peak File. Click OK.

When the program asks you whether to overwrite the entity, click OK.

This command reads in the peaks and displays them in a spreadsheet. The peaks are also displayed as boxes.

Activate the Peaks table and select File/Close to close it.

Select the second row in the Experiments table and click the Draw icon to display the TOCSY spectrum.

Activate the spectral window and select the File/Import/Peaks menu item. Set the Selection parameter to mcpn15tocsy.xpk and click OK.

When the program asks you whether to overwrite the entity, click OK.

Close the Peaks-xpk:tocsy table by selecting File/Close from its menu or by clicking the X button.

Select the third row in the Experiments table and click the Draw icon to display the noe spectrum.

Activate the spectral window and select the File/Import/Peaks menu item. Set the Selection parameter to mcpn15noe.xpk and click OK.

When the program asks you whether to overwrite the entity, click OK.

Close the Peaks-xpk:noe table.

Now you have a full peak set defined for all three experiments.

8.   Selecting the HSQC spectrum

Select the HSQC spectrum in the Experiments table and click the Draw icon.

Activate the spectral window. Press <Ctrl>-f to obtain the full plot if necessary.

The next step is the collection of prototype patterns, that is, sets of frequencies, which are later promoted to patterns and assigned to specific amino acid residues. The commands connected to prototype patterns are in the third subsection of the Assign pulldown. Since we have the 15N-HSQC, 15N-HMQC-TOCSY, and the 15N-HMQC-NOESY spectra in our project, we demonstrate the use of the two currently available double-resonance prototype pattern-collection methods.

9.   Performing prototype pattern detection

Select the Assign/Collect Prototype Patterns menu item. In the control panel, select Double Resonance for Method and click OK.

In the second control panel, set the Method to 3D HS(M)QC-TOCSY and click OK.

You see a control panel with several options. The program tries to automatically fill in reasonable values

.

Make sure these values are set in the third control panel:

Experiment tocsy
Root (Amide Proton) Dimension D1
Frequency Collapse Tolerance 0.08
Output Level Low

Now click More.... and set these values:

Seed Area D1 Low: 5.2
High: 12.0
Seed Area D2 Low: 5.2
High: 12.0
Seed Area D3 Low: 90
High: 130.0
Use Seed Peak D1 on
D2 off
D3 on
Exp. Area D1 Low: 5.2
High: 12.0
Exp. Area D2 Low: -2.0
High: 12.0
Exp. Area D3 Low: 90.0
High: 130.0
Use Exp. Peak D1 off
D2 on
D3 off
Remove Intraproto Frequencies on
Number of Frequencies in Proto Min 3
Max 12
Number of Iterations 8
Frequencies per Iteration 1

Leave the other parameters at their defaults and click OK.

Information about the current stage of prototype pattern collection appears in the output window. After one or two minutes, the prototype pattern collection finishes and a spreadsheet of prototype patterns is displayed. The following information appears in the output window:

     Nr of protos generated : ( 52)
The 3D protopattern detection took 4 seconds

The protein has 77 residues, from which you can theoretically expect only 71 spin systems to be found, since there are 5 prolines and the N- terminal spin system is probably missing. If you have recorded a well resolved 2D 15N-HSQC spectrum, then that can greatly help in spin- system collection. Therefore, we present here the other prototype pattern-detection method implemented in FELIX.

10.   Performing the second prototype pattern detection

Select the Assign/Collect Prototype Patterns menu item. In the control panel, select the Double Resonance option and click OK.

In the second control panel, select the 2D HSQC + 3D HS(M)QC-TOCSY option and click OK.

You get a third control panel with several options. The program tries to automatically fill in reasonable values.

Set these values in the third control panel:

Seed Experiment hsqc
Root Dimension D1
Expansion Experiment tocsy
Seed/Expansion Region Use Defaults
Frequency Collapse Tolerance 0.08
Output Level Low

Now click More... and set these values in the resulting control panel:

Seed Area D1 Low: 5.2
High: 12.0
Seed Area D2 Low: 90.0
High: 130.0
Use seed peak D1 on
D2: on
Expansion Area D1 Low: 5.2
High: 12.0
Exp. Area D2 Low: -2.0
High: 12.0
Exp. Area D3 Low: 90.0
High: 130.0
Use Exp. Peak D1 off
D2 on
D3 off
Remove Intraproto Frequencies on
Number of Frequencies in Proto Min 3
Max 12
Number of Iterations 8
Frequencies per Iteration 1

Leave the other parameters at their defaults and click OK.

Information about the current stage of prototype pattern collection appears in the output window. After one minute, the prototype pattern collection is finished, and the following information appears in the output window:

     Nr of protos generated : ( 13)
The 3D protopattern detection took 1 seconds

Now you have 65 prototype patterns in all. While this method relies on well resolved 2D HSQC peaks, the previous one depends on well resolved pseudo-diagonal peaks of the HMQC-TOCSY spectrum. In certain cases, the higher digital resolution and better-defined peak shapes of 2D spectra help find more spin systems, while in other cases relying on the third dimension yields better results. Sometimes the combination of the two is the best choice, as you can see from this example (the 15N-HSQC + 15N-HMQC-TOCSY would generate only 58 spin systems).

Since clearly some spin systems were missed, it is always advisable to inspect the peaks in the spectrum to see which ones were not assigned to spin systems. This procedure is shown in Step 13.

11.   Visually inspecting several prototype patterns

Select the tocsy experiment using the Experiment table: click the second row and then click the Draw icon. Select the third row in the Protopatterns table. Click the Zoom icon in the table.

The region (strip) containing peaks of the 3rd spin system is displayed.

Now connect the HSQC and HMQC-TOCSY spectra.

12.   Connecting the HSQC and TOCSY spectra

From the main menu select the Window/Add New Window menu item.

Activate the new frame (Frame 2) if necessary. Select the hsqc experiment in the Experiments table and click the Draw icon to display it in Frame 2.

Select the Preference/Frame Connection menu item. In the control panel, set First Frame to 2 and Second Frame to 1. Select Custom and click OK.

In the second control panel, set these values:

D1 D1
D2 Null
Define Jump on
D2 D3
Null Null

Click OK.

This connects the D1(1H) of HSQC with D1(1H) of HMQC-TOCSY, and D2(15N) of HSQC with D3(15N) of HMQC-TOCSY. If you switch to the frame containing the tocsy experiment, you can zoom in on the spectra (Zoom in Protopatterns table), but this time display the same region in both spectra.

You can use various methods to browse through the spectrum in Frame 2, and the same action occurs in Frame 1, too.

13.   Coloring the peaks based on prototype patterns

Activate Frame 2 and click the Full Plot icon to draw a full plot.

Select the Preference/Peak Display menu item. Set Coloring Mode to By Protos and leave the other parameters at their defaults. Click Draw.

A new control panel appears, where you can set the colors for peaks which have each frequency belonging to a prototype pattern (To The Same), and for those which do not (None).

Leave the values in the second control panel at their defaults and click OK.

From now on, when you use the View/Draw Peaks menu item, the peaks will be drawn according to this coloring scheme: green peak boxes will be drawn at peaks that belong to a prototype pattern, and red peak boxes will be drawn at peaks that were not assigned to any particular spin system. Therefore, the manual spin-system detection should proceed from those peaks which are, in this case, red.

When the full plot is drawn you can see that there are red and green peak boxes. You may notice a red peak box at the lower edge of the HSQC spectrum at around 124 ppm and 8.7 ppm.

Click the Zoom icon and zoom in on that peak. Then move the crosshair cursor on the center of that peak and click it.

This moves the display of the 3D TOCSY spectrum at that particular plane.

Now you learn to create a new spin system manually.

Make sure Frame 2 is the current frame. Select the Assign/Frequency Clipboard/Zero Clipboard menu item to clear the clipboard in Frame 2.

Now you are ready to add frequencies to this clipboard.

Select the Assign/Frequency Clipboard/Add One menu item.

When the crosshair cursor appears, click with it on the peak at 8.74, 124.1 ppm.

In the control panel, set Both 8.743666 124.1559 for Frequency and HN and N for Nucleus 1 and Nucleus 2 parameters, respectively and click OK.

Press the <Esc> key to quit the addition mode.

You can check what is in the clipboard by listing it (Assign/Frequency Clipboard/ View Clipboard), and the result is printed in the output window:

The Frequency Clipboard List contains the following frequencies:
     # 		Freq(ppm) 	Atom

--- --------- ----
1 8.744 H
2 124.156 N

Now switch to the frame containing the 3D TOCSY spectrum.

Click anywhere in Frame 2 to activate it. Click the Zoom icon and zoom in to the two visible peaks.

Select the Assign/Frequency Clipboard/Add One menu item. With the crosshair cursor, click the peak at around 8.74, 5.18 ppm.

No peak box drawn for this peak, because that peak was missed during peak picking since it is on the very edge of the spectrum.

In the control panel, set Frequency to F1_D2_H 5.182883 and Nucleus 1 to HX. Click OK.

With the crosshair cursor, click the peak at around 8.74 and 3.03.

In the control panel, set Frequency to F1_D2_H 3.031679 and Nucleus 1 to HX. Click OK.

Since this is the last frequency, press the <Esc> key to quit.

Since there were no peaks picked for these latter frequencies, your actual results may be different from the those presented. Check the clipboard again (Assign/Frequency Clipboard/View Clipboard):

The Clipboard List contains the following frequencies:
# Freq(ppm) Atom
     --- --------- ----
1 8.744 H
2 124.156 N
3 5.183 X
4 3.032 X

14.   Promoting the prototype patterns to patterns

Select the Assign/Promote Prototype Patterns menu item. In the first control panel, set the Copy parameter to Prototype Patterns to Spin Systems (Patterns) and click OK.

In the second control panel, do not change any values, just click OK.

After couple of seconds, 65 new patterns are generated and displayed in a spreadsheet. You can inspect the patterns using the Assign/Report Spin System menu item:

LISTING FOR PATTERN pa1

comment : fromproto1
color : Red
root frequency : 8.451

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7
8 9
hsqc tocsy noe null null null null null null
8.451 8.451 8.451 8.451 8.451 8.451 8.451 8.451 8.451 8.451 1:*_*:HN
123.444 123.444 123.444 123.444 123.444 123.444 123.444 123.444 123.444 123.444 1:*_*:N
3.739 3.739 3.739 3.739 3.739 3.739 3.739 3.739 3.739 3.739 1:*_*:HX
2.037 2.037 2.037 2.037 2.037 2.037 2.037 2.037 2.037 2.037 1:*_*:HX
0.816 0.816 0.816 0.816 0.816 0.816 0.816 0.816 0.816 0.816 1:*_*:HX

The neighbor probabilities
--------------------------
Pattern Probability
The residue type probabilities
------------------------------
Residue Probability

(i-1) Frequencies from protopattern 1
------------------------------------------

15.   Adding the manually detected spin system to the patterns

Select the Assign/Frequency Clipboard/Copy Clipboard to Pattern menu item. Keep the default parameters unchanged and click OK.

A new pattern with name pa66 is added to the database and is also displayed in the Spinsytsems table.

16.   Scoring the patterns

Select the Assign/Residue Type/Score Residue Type menu item. Set these values:

Use All Patterns On
Scoring Method All Atoms
Min Atoms N
Max Std Dev 3
Use COSY Peaks None
Database Store

Click OK.

After a few minutes, all 66 patterns are scored and the residue type probabilities are stored. Using the Assign/Report Spin System menu item for the first pattern will give similar results:

LISTING FOR PATTERN pa1

comment : fromproto1
color : Red
root frequency : 8.451

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
hsqc tocsy noe null null null null null null
8.451 8.451 8.451 8.451 8.451 8.451 8.451 8.451 8.451 8.451 1:*_*:HN
123.444 123.444 123.444 123.444 123.444 123.444 123.444 123.444 123.444 123.444 1:*_*:N
3.739 3.739 3.739 3.739 3.739 3.739 3.739 3.739 3.739 3.739 1:*_*:HX
2.037 2.037 2.037 2.037 2.037 2.037 2.037 2.037 2.037 2.037 1:*_*:HX
0.816 0.816 0.816 0.816 0.816 0.816 0.816 0.816 0.816 0.816 1:*_*:HX

The neighbor probabilities
--------------------------

Pattern Probability

The residue type probabilities
------------------------------

Residue Probability
ILE 1.000
VAL 1.000
THR 0.833
ARG 1.000
LYS 1.000
LEU 1.000

(i-1) Frequencies from protopattern 1
------------------------------------------

After the spin-system probabilities are defined, the next step is to find neighboring spin systems. This can be achieved here by using the 15N- HMQC-TOCSY spectrum. In such a spectrum you can expect cross peaks between the spins of the ith and (i+1)th residue, as well as between further separated residues. The algorithm should search for NOE cross peaks, such as HN,i-HN,i+1(-Ni+1), Ha,i-HN,i+1(-Ni+1), and Hb,i-HN,i+1(-Ni+1), whose presence makes the connectivity between the two spin systems probable. Before you start the neighbor search, the spectrum-specific shifts of the patterns for the 15N-HMQC-NOESY spectrum should be updated.

17.   Setting the spectrum-specific shifts for all patterns

Disconnect the frames by selecting the Preference/Frame Connection menu item and choosing the Disable option.

Select the NOE spectrum by selecting the Experiment/Select menu item in the Experiments table.

Since the chemical shifts in the patterns were defined using the HSQC and the 15N-HMQC-TOCSY spectrum, a slight difference is expected between those shifts and the actual shifts in the 15N-HMQC-NOESY spectrum. To take into account this possible shift difference, you need to edit the spectrum-specific shifts of the patterns. This can be done either manually (where for each pattern the chemical shifts of frequencies are adjusted based on displayed intrapattern peaks) or automatically.

Select the Assign/Spin System/Auto Update Specific Shifts menu item. Select the noe spectrum and click All. Set the tolerances to 0.02, 0.04 and 0.1. Click OK.

In few minutes the spectrum-specific chemical shifts are set for all the patterns. You can see the results by using the Assign/Report Spin System menu item for e.g. the first pattern:

LISTING FOR PATTERN pa1

comment : fromproto1
color : Red
root frequency : 8.451

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
hsqc tocsy noe null null null null null null
8.451 8.451 8.451 8.446 8.451 8.451 8.451 8.451 8.451 8.451 1:*_*:HN
123.444 123.444 123.444 123.447 123.444 123.444 123.444 123.444 123.444 123.444 1:*_*:N
3.739 3.739 3.739 3.734 3.739 3.739 3.739 3.739 3.739 3.739 1:*_*:HX
2.037 2.037 2.037 2.023 2.037 2.037 2.037 2.037 2.037 2.037 1:*_*:HX
0.816 0.816 0.816 0.803 0.816 0.816 0.816 0.816 0.816 0.816 1:*_*:HX

The neighbor probabilities
--------------------------

Pattern Probability

The residue type probabilities
------------------------------

Residue Probability
ILE 1.000
VAL 1.000
THR 0.833
ARG 1.000
LYS 1.000
LEU 1.000

(i-1) Frequencies from protopattern 1
------------------------------------------

You must update the root frequency attribute of the patterns:

Select the Assign/Spin System/Copy Specific Shift to Generic menu item. Set Patterns to All and Spectrum to NOE. Click OK.

Now select the Assign/Spin System/Auto Root menu item, set Patterns to All, and click OK.

18.   Performing neighbor searches

Select the Assign/Neighbor/Find Neighbor Via 3D N-15 NOE menu item. Set these values:

Experiment noe
Candidate (NOE) Dimension D2
Frequency Collapse Tolerance 0.04
Specify Patterns All Patterns
Normalize Scores on
Store Scores Overwrite Old
Number of Neighbors to Save 12
Output Level Low

Click More... and set these values:

Number of Frequencies to Use in Pattern 6
First Type H
High 12.0
Low 6.0
Second Type N
High 130.0
Low 90.0
Candidate Frequency Range
High 12.0
Low -2.0
Minimum # of NOE Peaks 2

Click OK.

In few seconds the neighbor search is done. There are several ways to check for the result of the run; using the previously described Assign/ Report Spin System menu item now will result in output such as:

LISTING FOR PATTERN pa1

comment : fromproto1
color : Red
root frequency : 8.446

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
hsqc tocsy noe null null null null null null
8.446 8.451 8.451 8.446 8.451 8.451 8.451 8.451 8.451 8.451 1:*_*:HN
123.447 123.444 123.444 123.447 123.444 123.444 123.444 123.444 123.444 123.444 1:*_*:N
3.734 3.739 3.739 3.734 3.739 3.739 3.739 3.739 3.739 3.739 1:*_*:HX
2.023 2.037 2.037 2.023 2.037 2.037 2.037 2.037 2.037 2.037 1:*_*:HX
0.803 0.816 0.816 0.803 0.816 0.816 0.816 0.816 0.816 0.816 1:*_*:HX

The neighbor probabilities
--------------------------

Pattern Probability
pa12 0.2500
pa28 0.1875
pa51 0.1875
pa48 0.1250
pa50 0.1250
pa62 0.1250

The residue type probabilities
------------------------------

Residue Probability
ILE 1.000
VAL 1.000
THR 0.833
ARG 1.000
LYS 1.000
LEU 1.000

(i-1) Frequencies from protopattern 1
------------------------------------------

You can also use the Assign/Neighbor/List Neighbors menu item:

Select the Assign/Neighbor/List Neighbors menu item. Select pa1 from the List of Patterns and leave Order set to i - i+1. Click OK.

The output will be similar to:

     The possible neighbors (i - i+1) for pattern pa1 are:
pattern pa12 with probability: 0.2500
pattern pa28 with probability: 0.1875
pattern pa51 with probability: 0.1875
pattern pa48 with probability: 0.1250
pattern pa50 with probability: 0.1250
pattern pa62 with probability: 0.1250

Or you can visually inspect the neighboring patterns:

Go to the Spinsystems table and select the first pattern by clicking it. Then select the Spinsystem/Show i+1 Neighbors Via Strip Plot menu item.

Seven strips appear on the screen, containing plots of the region containing the frequencies of pattern pa1, and the neighboring patterns: pa12, pa28, pa48, pa50, pa51, and pa62. Also, the output window shows:

     Strip plot of pattern 1 with neighbors:
pa12 0.2500
pa28 0.1875
pa51 0.1875
pa48 0.1250
pa50 0.1250
pa62 0.1250

The next step is to generate possible sequence-specific assignments for the patterns, that is, to compare spin-system type and neighbor-probability information with the primary sequence and make suggestions about which pattern belongs to which particular amino acid in the sequence. This can be done in FELIX through the Assign/Sequential menu items. Here we show one approach, using the Assign/ Sequential/ Systematic Search menu item; other approaches can be found in the Tasks Chapter of the separate FELIX User Guide.

Since the 15N-HMQC-TOCSY spectrum does not contain spin systems from prolines therefore we need to find stretches of sequential assignments between the Pro residues. The first such one in the sequence is between residues 4 and 8. Certainly, if one has the Pro spin systems detected, then this limitation does not exist.

19.   Generating sequence-specific assignments for the stretch of residues 4-8

Select the Assign/Sequential/Systematic Search menu item and set these values:

Min individual assignment prob 0.8
Min Neighbor Prob Score 0.15
First residue (#) to consider 4
Last residue (#) to consider 8
Min Length of Assigned Stretches 4
Output level Low

Leave the other parameters at the default values and click OK.

After few seconds you will see a listing of several possible assignments, the beginning of which would look like:

Sorting out and storing the generated assignments
Assignments left :( 8 )

assignment # 1 -- length = 5 residues
... stretch of residues = 4 - 8 total scores : 4.95 1.83
Residues : ASP-_4 ALA_5 ILE_6 ASN_7 ALA_8
Patterns : 36 37 4 44 58
Scores : 0.99 0.99 0.99 0.99 0.99
I>I+1 : 0.33 0.50 0.33 0.67

assignment # 2 -- length = 4 residues
... stretch of residues = 5 - 8 total scores : 3.96 2.00
Residues : ALA_5 ILE_6 ASN_7 ALA_8
Patterns : 33 4 44 58
Scores : 0.99 0.99 0.99 0.99
I>I+1 : 1.00 0.33 0.67

...
The Pattern Suggest Assignment took 2 seconds!

Also, a new spreadsheet is activated: Stretches where stretches of possible sequential assignment are stored.

20.   Showing the first possible sequential assignment

Now you can inspect the first possible stretch.

Go to the Stretches table and select the first row by clicking it, then click the ND Strip Plot icon.

In few seconds a strip plot appears which contains five vertical strips -  along the HN-N frequencies of the patterns pa36, pa37, pa4, pa44, and pa58. Also, a message is printed in the output window:

     . Strip plot of stretch # 1
The patterns in the stretch are:
pa36
pa38
pa4
pa44
pa58

21.   Generating sequence-specific assignments for the stretch of residues 4-8 via simulated annealing

Next you try the other method available for sequential assignment: simulated annealing.

Select the Assign/Sequential/Simulated Annealing menu item. Set these values:

Min Individual Assignment prob 0.8
First Residue (#) to Consider 4
Last Residue (#) to Consider 8
Discard Previous Assignments False
Store Generated Assignments False
Output Level High

Leave the other parameters at the default value (1.0) and click OK.

The output in the output window should be similar to this:

SA completed: initial energy 6.500 and final energy 2.000(niter 30)
new sequence (energy 1.875):
10 33 4 44 58

Suggested assignment:
ASP4 -> pa10 Type score 1.000 Neighbor probability 0.125
ALA5 -> pa33 Type score 1.000 Neighbor probability 1.000
ILE6 -> pa4 Type score 1.000 Neighbor probability 0.333
ASN7 -> pa44 Type score 1.000 Neighbor probability 0.667
ALA8 -> pa58 Type score 1.000
The Pattern Suggest Assignment took 3 seconds!

Now you can inspect this assignment too.

Go to the Spinsystems table and first select the tenth row (pa10). Then <Ctrl>-click to select the rows containing pa33, pa4, pa44, and pa58.

Click the ND Strip Plot icon.

In the output window a message appears:

      Strip plot of pattern 10
Strip plot of pattern 33
Strip plot of pattern 4
Strip plot of pattern 44
Strip plot of pattern 58

and five vertical strips are displayed in the frame.This solution was not found in systematic search, since the neighbor probability measure between pa10 and pa33 is 0.125, which is lower than the cutoff we set (0.15).

Using the simulated annealing and systematic search methods for sequential assignment one can then assign the patterns to specific residues.

22.   Exiting FELIX

To exit FELIX, select the File/Exit menu item.


Lesson 3: Heteronuclear triple resonance 3D assignment strategy

This lesson takes you through typical steps in the assignment of a doubly-labelled protein. The data set consists of the HNCACB and CBCA(CO)NH spectra of the 13C- and 15N-enriched RNA-binding domain of hnRNP C from Luciano Mueller (Bristol-Myers Squibb, Princeton: see Wittekind 1992).

The topics covered in this lesson are:

1.   Setting up for the lesson

If not done yet, set up the tutorial files as described in "Setting up tutorial files" in the preface, How to use this book.

The files for this lesson are all located in the Assignment\Lesson3 folder

2.   Starting FELIX

Start FELIX by double-clicking the FELIX icon on your desktop, or by clicking the Start button on the Windows taskbar, then selecting Programs/Accelrys FELIX 2004/FELIX 2004.

If FELIX prompts you to restore from last session, click Cancel.

Change your Current Working Directory to C:\Felix_Practice\Assign\Lesson3\ using the Preference/Directory... command. Build a new database by opening the File/New... command and choosing Create a new matrix or DBA file. Make sure the File Type is set to DBA(*.dba). Enter hnrp.dba and click OK.

3.   Setting up the database

Select the Assign/Project menu item. When a dialog box appears informing you that no project was found in the database and asking if you want to build one, click OK.

In the next control panel, enter a name (e.g., rnp:project).

In the next control panel you need to select a molecule, in this case an extended chain of hnRNP C:

Selection hnrnp.car
File Type Insight Molecule (*.car)

Click OK to build the entities and read in the molecule.

This procedure typically takes several seconds; a meter on the status bar shows the progress.

4.   Defining a library

After the setup is successfully completed, you should define a library. The library is an ASCII file, as described in the Assign/Define Library section of the separate FELIX User Guide. FELIX contains a standard library (pd.rdb) which you should read in.

In the next control panel (Library) Select Define Library from File. In the next control panel, select pd.rdb, which stands for protein-DNA library. A few seconds later the setup procedure is finished.

5.   Adding experiments to the projects

Select the Assign/Experiment menu item to define new experiments in the assignment database. When the control panel appears with names of matrices, select hncacb.mat (the HNCACB spectrum).

Click OK.

Set these parameter values for the plot using the 3D DISPLAY PARAMETERS control panel:

Contour Threshold 0.3
Color Scheme Blue/Green
Axis type D1 ppm
Axis type D2 ppm
Axis type D3 ppm
D1 Scale 1
D2 Scale 1
D3 Scale 1

Leave the other parameters at their default values and select Apply.

Click OK when the message box appears.

If you want, you can change the display parameters using the Experiment/Change Attribute menu item in the Experiment table.

The program plots a density plot or contour plot of the HNCACB using the parameters you defined. Note, since by default this is the first plane, that possibly no cross peaks may be seen, but you can select a different plane (or different view) using the New Plane button later.

Now another control panel appears.

Set the parameters to these values:

Title hnccb

Use Default Names on
Type 3D CBCANH
Temperature 298
pH 7
Solvent Water
D1 Nucleus Carbon
D2 Nucleus Nitrogen
D3 Nucleus Proton
D1 Tol 0.15
D2 Tol 0.15
D3 Tol 0.02
D1 Fold No
D2 Fold No
D3 Fold No
W1 D3
W2 D2
W3 D1
W1-W2 Transfer J-coupled
# of J Steps 1
W2-W3 Transfer J-coupled
# of J Steps 1

Click OK.

It is important to define the spectrum-specific tolerances, since they are used in many automated and semi-automated procedures.

A new window containing an Experiments table is open and displayed to the left of the spectral window. Note that, by default, whenever a new window (table or spectral) is open, FELIX automatically re-arranges the layout of the windows. You can turn off this feature by selecting Preference/Frame Layout from the main menu and set Action to None. You can also do the automatic re-arrangement at anytime by selecting Window/Auto Arrange.

Note: When one or more table windows are open, only the menu and tool bar of the currently activated window are visible. If you want to select a certain menu item or tool bar icon, be sure to click the corresponding window first to activate its menu and tool bar (if any).

6.   Adding the CBCA(CO)NHN spectrum to the project

Activate the Experiments table. Select the Experiments/Add menu item.

Select cbcaconh.mat and set these values:

Contour Threshold 0.3
Color Scheme Magenta
Number of Levels 16
Axis Type ppm
Negative Levels Off.

Click OK.

In the next control panel, set the parameters to these values:

Title cacbconh

Use Default Names on
Type 3D CBCA(CO)NH
Temperature 298
pH 7
Solvent Water
D1 Nucleus Carbon
D2 Nucleus Nitrogen
D3 Nucleus Proton
D1 Tol 0.15
D2 Tol 0.15
D3 Tol 0.02
D1 Fold No
D2 Fold No
D3 Fold No
W1 D3
W2 D2
W3 D1
W1-W2 Transfer J-coupled# of J
Steps 1
W2-W3 Transfer J-coupled # of J
Steps 1

Click OK.

7.   Reading in the peaks

In the Experiments table select the hncacb experiment (click the first row) and click the Draw icon. This draws the hncacb spectrum in the spectral frame.

Activate the spectral frame and select the File/Import/Peaks menu item. Select hncacb.xpk and leave the FELIX Peak Table Name parameter at its current status (xpk:hncacb). Click OK.

When the query box asks you about overwriting the entity, click OK.

This action reads in the peaks and also displays them as a peak table.

Now select the cbcaconh experiment in the Experiments table and click the Draw icon to plot it.

Activate the spectral window and select the File/Import/Peaks menu item. Select cbcaconh.xpk and click OK. In the new query box asking about overwriting the entity, click OK.

Now you have a full peak set defined for both experiments. Close the two peak tables by selecting File/Close from their menus.

8.   Selecting the HNCACB spectrum

Now reselect the hncacb experiment from the Experiments table and click the Draw icon to display the spectral window.

The next step is the collection of prototype patterns, i.e., sets of frequencies, which later are promoted to patterns and assigned to specific amino acid residues. The commands related to prototype patterns are in the Assign pulldown in the third subsection. Since we have the HNCACB and the CBCA(CO)NH spectra in our project, we demonstrate the use of one of the triple resonance prototype pattern collection methods.

9.   Performing a prototype pattern detection

Activate the spectral window and select the Assign/Collect Prototype Patterns menu item. In the control panel, select the Triple Resonance option and click OK.

In the following control panel, select the CBCANH + CBCA(CO)NH option and click OK.

Set these parameter values in the third control panel:

Use Default Experiments on
CA peak intensity sign +
CB peak intensity sign -

CA peak intensity sign +
CB peak intensity sign +

Tolerances
HN
0.02
C 0.2
N 0.2
Number of iterations 5
Tolerance factor 1.2
Output level Low

Click OK.

After a couple of minutes, the prototype pattern collection is finished, and the following information appears in the output window:

The 3D protopattern detection took 154 seconds for 5 iteration
and the total number of prototype patterns now : 79
The number of unclassified peaks in cbcaconh.mat is: 134
The number of unclassified peaks in hncacb.mat is: 231

A new table of prototype patterns also appears. Comparing these prototype patterns with the spin systems assigned previously (1) we confirm that 82% of the spin systems were picked correctly.

10.   Visually inspecting several prototype patterns

From the Protopatterns table select the first protopattern and then click the Zoom icon.

The region containing the four HNCACB peaks of the first protopattern is displayed.

In order to see the HNCACB and CBCACONH peaks simultaneously when you browse the prototypes, you use two connected spectral windows as follows.

11.   Connecting the HNCACB and CBCACONH spectra

For the following action you need to have room for two spectral frames.

Activate the spectral window and select the Window/New Frame Layout menu item. In the control panel, set the New Layout parameter to 2 Frames Up/Down. Click OK.

Two spectral frames are displayed and the other table windows are re- organized automatically. Since the up/down layout of two spectral windows is not the default layout of FELIX, you have to toggle off the automatic rearrangement option in order not to mess up your layout when a new window

Select the Preference/Frame Layout menu item. Set Action as None for Auto. Frame Organization. Select OK.

Now activate Frame 2 and then select the cbcaconh experiment in the Experiments table. Click the Draw icon.

Next activate Frame 1 and click the Plot icon.

While Frame 1 is activated, select the Preference/Frame Connection menu item. In the control panel, leave 1 for the First Frame and 2 for the Second Frame parameters. Select D1-D2-D3 <=> D1-D2-D3 and click OK.

Now the two spectra are connected in all three dimensions. Now you can navigate in the first frame and the display is updated in the second frame, too. For example, if you zoom on a prototype pattern in the first frame, the same region is displayed in the second frame, too.

You can use different activities to browse through the spectrum in Frame 1, and the same action also takes place for Frame 2.

12.   Correcting the prototype patterns

In this section you find out how to make corrections to the automatically detected prototype patterns. To do this, you need to zoom on a prototype pattern and then inspect the frequencies (which are drawn through the peaks).

Go to the Protopatterns table, select the first prototype pattern, and click the Zoom icon.

Alternatively, you can double-click the first row, since the default action for double-clicking is to zoom. You can change the default action using the popup on the combo box of the table.

You can see that the first prototype pattern is correct. Now zoom on the second prototype pattern, and you can see that this one is also correct. When you zoom on the third prototype pattern, you can see that only three carbon frequencies were found and that there is a well-defined peak at 64.1, 121.8, 9.4 ppm. If you look at the prototype pattern in the table you can see that the CA(i) was missed, and most likely this peak contains that frequency (64.1 ppm). Here, you will add that frequency:

Go to the Protopatterns table and select the ProtoPattern/Add Frequency via Cursor menu item.

With the crosshair cursor, click the CA(i) peak that was missed. In the ADD FREQUENCY TO PROTOPATTERN 3 control panel, set the Frequency to F1 64.06 and the Nucleus 1 to CA. Click OK.

Now the prototype pattern is redrawn on the screen with four carbon frequencies and the table is updated: there is now a CA frequency for the third protopattern.

You would typically go through the full set of prototype patterns and make similar adjustments. Also, you can edit the frequencies in the table directly or by using the Assign/Edit Prototype menu item. You can also delete spurious protopatterns through the table or through the Assign/ Edit Prototype menu item.

After the prototype patterns are cleaned up you can promote them to patterns.

13.   Promoting the prototype patterns to patterns

Select the Assign/Promote Prototype Patterns menu item. In the first control panel, select the Copy Prototype Patterns to Spin Systems (Patterns) option and click OK.

In the next control panel, set the Sequential Tolerances for C to 0.25, set the Find Prolines to Yes, and leave the Compare Similarities parameter as No. Click OK.

You can follow the progress in the output window: in first stage you can see that the regular spin systems are to be promoted - i.e., non-prolines. Then FELIX tries to find spin systems that can be interpreted as prolines - i.e., such protopatterns that contain a Ca,i-1 in the 60-66 ppm range and a Cb,i-1 in the 28-34 ppm range and no appropriate HN and N. In third stage FELIX tries to find the possible sequential connectivities and stores them with the newly generated patterns as probabilities. After a few minutes 86 new patterns are generated and the table containing them is displayed (Spinsystems). You can either inspect the patterns in the table or use the Assign/Report Spin System menu item:

LISTING FOR PATTERN pa1
comment : fromproto1
color : Red
root frequency : 9.637

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
hncacb cbcaconh null null null null null null null
9.637 9.637 9.637 9.637 9.637 9.637 9.637 9.637 9.637 9.637 1:*_*:HN
129.947 129.947 129.947 129.947 129.947 129.947 129.947 129.947 129.947 129.947 1:*_*:N
61.505 61.505 61.505 61.505 61.505 61.505 61.505 61.505 61.505 61.505 1:*_*:CA
34.436 34.436 34.436 34.436 34.436 34.436 34.436 34.436 34.436 34.436 1:*_*:CB

The neighbor probabilities
--------------------------

Pattern Probability
pa4 0.3333
pa37 0.2222
pa38 0.2222
pa70 0.2222

The residue type probabilities
------------------------------

Residue Probability

(i-1) Frequencies from protopattern 1
------------------------------------------

CA(i-1) 54.484 ppm
CB(i-1) 33.110 ppm

14.   Scoring the patterns

Select the Assign/Residue Type/Score Residue Type menu item. Check Use All Pattern. Set the Scoring Method to CACB only. Select Store for Database. Click OK.

After few seconds all 86 patterns are scored and the residue type probabilities are stored. Using the Assign/Report Spin System menu item for the first pattern gives a similar result:

LISTING FOR PATTERN pa1

comment : fromproto1
color : Red
root frequency : 9.637

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
hncacb cbcaconh null null null null null null null
9.637 9.637 9.637 9.637 9.637 9.637 9.637 9.637 9.637 9.637 1:*_*:HN
129.947 129.947 129.947 129.947 129.947 129.947 129.947 129.947 129.947 129.947 1:*_*:N
61.505 61.505 61.505 61.505 61.505 61.505 61.505 61.505 61.505 61.505 1:*_*:CA
34.436 34.436 34.436 34.436 34.436 34.436 34.436 34.436 34.436 34.436 1:*_*:CB

The neighbor probabilities
--------------------------

Pattern Probability
pa4 0.3333
pa37 0.2222
pa38 0.2222
pa70 0.2222

The residue type probabilities
------------------------------

Residue Probability
MET 0.001
VAL 0.479
LYS 0.009
PRO 0.471
PHE 0.001
ILE 0.037
TYR 0.002

(i-1) Frequencies from protopattern 1
------------------------------------------

CA(i-1) 54.484 ppm
CB(i-1) 33.110 ppm

15.   Generating sequence-specific assignments for patterns

Select the Assign/Sequential/Systematic Search menu item. Set the Min Individual Assignment Prob to 0.1 and set the Last residue (#) to Consider to 94. Set the Min Length of Assigned Stretches to 3. Select Low for Output level and leave the other parameters at the default value. Click OK.

After few seconds you will see many possible assignments; the beginning of the output will look like:

assignment # 1 -- length = 10 residues
... stretch of residues = 69 - 78 total scores : 5.38 3.74
Residues : GLY_69 GLU-_70 ASP-_71 GLY_72 ARG+_73 MET_74 ILE_75 ALA_76
Patterns : 49 64 58 41 77 20 22 2
Scores : 0.99 0.17 0.26 0.99 0.23 0.19 0.43 0.99
I>I+1 : 1.00 0.27 0.43 0.33 0.33 0.33 0.38 0.33
Residues : GLY_77 GLN_78
Patterns : 31 76
Scores : 0.99 0.14
I>I+1 : 0.33

assignment # 2 -- length = 9 residues
... stretch of residues = 70 - 78 total scores : 4.37 2.80
Residues : GLU-_70 ASP-_71 GLY_72 ARG+_73 MET_74 ILE_75 ALA_76 GLY_77
Patterns : 55 58 41 77 20 22 2 31
Scores : 0.13 0.26 0.99 0.23 0.19 0.43 0.99 0.99
I>I+1 : 0.33 0.43 0.33 0.33 0.33 0.38 0.33 0.33
Residues : GLN_78
Patterns : 64
Scores : 0.16

assignment # 3 -- length = 9 residues
... stretch of residues = 70 - 78 total scores : 4.35 2.80
Residues : GLU-_70 ASP-_71 GLY_72 ARG+_73 MET_74 ILE_75 ALA_76 GLY_77
Patterns : 55 58 41 77 20 22 2 31
Scores : 0.13 0.26 0.99 0.23 0.19 0.43 0.99 0.99
I>I+1 : 0.33 0.43 0.33 0.33 0.33 0.38 0.33 0.33
Residues : GLN_78
Patterns : 76
Scores : 0.14

The scoring stores the results in the database as stretches. At the end of the action the stretches are shown in tabular form. This table can be closed and reopened using the Edit/Stretch menu item. Next, we show how to use stretches in helping with the assignment procedure.

16.   Reviewing the first stretch

First increase the size of the frame containing the hncacb experiment (Frame 1). Now go to the Stretches table and select the first row by clicking it, then click the ND Strip Plot icon.

You will see the strip plot of patterns 49, 64, 58, 41, 77, 20, 22, 31, and 7. I

17.   Setting the sequence-specific assignments for patterns 49, 64, 58, 41, 77, 20, 22, 2, 31, and 76

Go to the Stretches table. With the first row highlighted, select the Stretch/Assign One Stretch menu item. Start the stretch at GLY_69 and leave the Assign Frequencies on. Click OK.

During execution the following lines appear in the output window:

      Pattern pa49 assigned to residue 1:GLY_69.
Pattern pa64 assigned to residue 1:GLU-_70.
Pattern pa58 assigned to residue 1:ASP-_71.
Pattern pa41 assigned to residue 1:GLY_72.
Pattern pa77 assigned to residue 1:ARG+_73.
Pattern pa20 assigned to residue 1:MET_74.
Pattern pa22 assigned to residue 1:ILE_75.
Pattern pa2 assigned to residue 1:ALA_76.
Pattern pa31 assigned to residue 1:GLY_77.
Pattern pa76 assigned to residue 1:GLN_78.

When you are done you can check the resulting pattern using the Assign/Report Spin System menu item. For example, pattern 58 would yield a similar result:

LISTING FOR PATTERN pa58

comment : fromproto58
color : Red
root frequency : 7.883

Frequencies
-----------
generic specific assignment
shift shifts
1 2 3 4 5 6 7 8 9
hncacb cbcaconh null null null null null null null
7.883 7.883 7.883 7.883 7.883 7.883 7.883 7.883 7.883 7.883 1:ASP-_71:HN
122.905 122.905 122.905 122.905 122.905 122.905 122.905 122.905 122.905 122.905 1:ASP-_71:N
57.338 57.338 57.338 57.338 57.338 57.338 57.338 57.338 57.338 57.338 1:ASP-_71:CA
41.030 41.030 41.030 41.030 41.030 41.030 41.030 41.030 41.030 41.030 1:ASP-_71:CB

The neighbor probabilities
--------------------------

Pattern Probability
pa3 0.1429
pa18 0.2857
pa41 0.4286
pa72 0.1429

The residue type probabilities
------------------------------

Residue Probability
ASP 0.257
PHE 0.244
ILE 0.050
LEU 0.214
TYR 0.235

(i-1) Frequencies from protopattern 58
------------------------------------------

CA(i-1) 54.775 ppm
CB(i-1) 30.755 ppm

18.   Exiting FELIX

To exit FELIX, select the File/Exit menu item.