| Felix Tutorials |
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.
This lesson presents the basic steps of an NMR spectrum assignment. In this lesson you use prepared data matrices: namely zh.mat, zc.mat, zn.mat, and zb.mat. These are TOCSY, DQF-COSY, and NOESY 2D NMR spectra, respectively, of Zn-rubredoxin (Blake et al. 1991). The molecular coordinates are also available in the file $BIOSYM/tutorial/felix/znrdlec.car.
The topics covered in this lesson are:
1. Setting up for the lesson
2. Starting Felix
3. Going to the Assign module
Select the Assign/Project item from the menubar.
4. Setting up the database
In the next control panel, select the linear chain of the molecule by setting Selection to znrdlec.car.
Select 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 under Assign in the 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 select 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 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.
Now another control panel appears.
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.
|
In the next control panel, set these parameter values for TOCSY:
Parameter Name TOCSY Value NOESY Value Experiment Title tocsy noe |
|
In the next control panel set these values:
Parameter Name TOCSY Value NOESY Value Type 2D TOCSY 2D NOESY |
8. Checking the project entity
Check the project entity after all experiments are added as described in Step 5.
Note that previously zero or null fields now have values.
9. Drawing the full DQF-COSY spectrum
Now go to the Experiments table and select the cosy spectrum by clicking the first row and then clicking the Select Experiment icon.
Now click the Full Plot icon to draw the full DQF-COSY spectrum.
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 Felix User Guide), which also increases the accuracy of peak picking. 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, we have made an attempt to provide you with 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
When the query box appears, asking about overwriting the entity, select OK.
Go to the Experiment table and select the TOCSY spectrum as described above.
Select the View/Plot menu item to plot the spectrum.
Next you repeat the procedure for the NOE spectrum.
Select the NOE spectrum through the Experiments table.
Plot the NOE spectrum by selecting the View/Plot menu item.
Now you have a full peak set defined for all three experiments.
11. Selecting the DQF-COSY spectrum
Select the DQF-COSY spectrum using the Experiments table. Press <Ctrl>-f on your keyboard to obtain the full plot.
Those footprints were drawn that 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 select 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. Select OK.
A control panel with several options appears. The program tries to fill in reasonable values.
The Frequency Collapse Tolerance is the tolerance for aligning and finding connected expansion peaks with seed peaks.
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.
The expansion area will cover the full spectrum. # ppm filters active 2 Filter # 2
In the remaining part of the control panel, set these parameter values:
1
Low 6
High 12
Min 1
Max 1
Low 3
High 5.5
Min 1
Max 3
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 text 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 text window:
Nr of prototype patterns generated:(57)
The 2D protopattern detection took 37 seconds
Also, a spreadsheet containing the prototype patterns is displayed (Protopatterns).
13. Saving the results of prototype pattern detection as a file
Go to the table and select the File/Save As menu item. Set the Selection to zn_protos.txt and select OK.
In the text window you are informed about the success of the command:
Wrote table: zn:proto
Created file: ./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.
You see four lines at 9.7, 5.37, 1.78, and 0.89 ppm, which are frequencies in this prototype pattern.
Right-click the Clear Frequencies control.
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.
Press <Ctrl>-c (if you were in intensity-plot mode) to see the magenta contour plot of the TOCSY spectrum tiled by the first prototype pattern.
You can also display frequencies by clicking the Draw icon in the table.
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 Shift Type Generic
Press <5> 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:
Dimension W2
Width 64
Scale 4.
You see four vertical strips with the frequencies of the first prototype pattern in the middle of each. You can also display frequencies by selecting the Draw icon.
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
Select the Assign/Frequency Clipboard/Copy Proto to Clipboard menu item. In the control panel, select 1 from the List of Protos and select 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. |
You can see the sorted clipboard by selecting the Assign/Frequency Clipboard/View Clipboard menu item. 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.
To copy the clipboard list to a pattern, a pattern must already exist.
Select the Assign/Spin System/Add One menu item. Leave pa1 as the Name and enter 9.703 as the Root freq. Select OK.
This action creates a new pattern. Now copy the frequency clipboard to this pattern. Alternatively, you can create the new pattern while copying (Assign/Frequency Clipboard/Copy Clipboard to Pattern), by setting the Mode parameter to New.
18. Copying the frequency clipboard to the pattern
Select the Assign/Frequency Clipboard /Copy Clipboard to Pattern menu item. In the control panel, select the only existing pattern (pa1) for Mode parameter Append and select OK.
Now you have a new pattern with four frequencies. Also, a new spreadsheet is displayed--Spinsystems. You can list this pattern to a file or to the text 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.
19. Viewing the pattern
Select the Assign/Report Spin System menu item. Click pa1 and make sure that Action is set to To Textport and Specific is selected as the Patterns parameter. Select OK.
This prints the following information to the text 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.
20. Finding the residues that the pattern belongs to
In the text 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.
21. 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. Select OK.
The result is a table in the text 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 H
is the frequency with 5.369 ppm. H
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.
22. Assigning the frequencies
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.
23. 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. Select OK.
You see the spectrum-specific shifts drawn on top of the tiled plot of the NOE spectrum, and a message in the text window:
Pick on the frequency to edit!
Use the crosshair cursor to click the frequency you want to edit:
| Put the large crosshair cursor on top of the frequencies at 9.703 and 0.895 ppm in D1 and D2, respectively and click the left mouse button. |
A new message appears in the textport:
Pick on the new position!
| Put the large crosshair cursor on a position that is closer to the peak box center (better aligned) and click the left mouse button. |
The new shifts are displayed in green, and a message appears with the new chemical shifts. You may have something similar to:
9.70447 0.89068
| Repeat these steps for each frequency to obtain the best alignment. |
| 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. |
The results should be similar to these:
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 9.702 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 1.794 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 0.892 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
You now need to inspect the other prototype patterns and promote them to patterns, as was done in Steps 16, 17, and 18.
24. Copying the 55th prototype pattern to the clipboard list
You can see that, in the column of 9.194 ppm, there is an extra frequency around 2.5 ppm.
Score the pattern as in Step 20. 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, 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 22. 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
25. 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 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. Match against it, and then assign it. |
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 lysine 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
26. Copying the 49th prototype pattern to the frequency clipboard
Follow the procedure described in Step 26 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 peak to the left of the peak 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.
You see the following listing in the text window:
The probability for pa4 to be LYS is : 1.000
The probability for pa4 to be VAL is : 0.833
The probability for pa4 to be ILE is : 0.714
The probability for pa4 to be PRO is : 0.714
The probability for pa4 to be LEU is : 1.000
| 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 select 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 0.853 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
0.853 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
27. Copying the 4th prototype pattern to clipboard list
This time you will promote (copy) a prototype pattern directly to a pattern (spin system).
The residue type scoring appears in the text 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.
28. 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. 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. Also, make sure to select the NOE spectrum from the Experiments table.
After one or two seconds the results are printed and stored:
Spectrum :(zn.mat) Tolers :( 0.010 0.010 )
Pattern :(ALL)
Now at pattern :( 1 )
Use :( 5.369 1.786 0.895 )
Patterns :( 2 )
Scores :( 1.000 )
Now at pattern :( 2 )
Use :( 3.060 2.845 2.665 )
Patterns :( 3 )
Scores :( 1.000 )
Now at pattern :( 3 )
Use :( 3.972 1.983 1.498 1.674 )
Patterns :( 4 )
Scores :( 1.000 )
Now at pattern :( 4 )
Use :( 4.049 2.545 1.366 0.846 )
Patterns :( 5 )
Scores :( 1.000 )
Now at pattern :( 5 )
Use :( 5.022 3.288 2.526 ) No candidate neighbours
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.
In the text window you see:
The possible neighbors for pattern pa1 are:
pattern pa2 with probability: 1.0000
Similarly for pa2, pa3, and pa4:
The possible neighbors for pattern pa2 are:
pattern pa3 with probability: 1.0000 The possible neighbors for pattern pa3 are:
pattern pa4 with probability: 1.0000 The possible neighbors for pattern pa4 are:
pattern pa5 with probability: 1.0000
29. 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 (dH(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.
30. Matching the found patterns against the known amino acid sequence
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 0.000 0.000 1.000 )
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 4.00
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 1.00
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.
31. 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 22, you next de-assign the non-sequence-specific assignment for each frequency and make the sequence-specific ones.
Click 9.712 ppm in Frequency list and then click Select.
Select 1:VAL_*:HN from the Assignments list and click Delete, since you want to reassign this frequency.
(You may skip the de-assignment, since the following step automatically sets the assignment pointers.)
Now select VAL from the Residues list, 4 from the #'s list, and HN from the Protons list.
Click Build.
| 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 the only item in the Assignments list. This puts that atom name into the Atom Spec box. Next you can edit that string, replacing the * with 4, and then click Add. When the two atom specs show up in the list, you can select 1:VAL_*:HA and click Delete. You can then repeat this procedure for each frequency in this pattern.
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.
32. 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. Fully assigned Green
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:
Partially (D1) assigned Magenta
Partially (D2) assigned Cyan
Multiply assigned Blue
Not assigned Red
All the peaks should now be red, indicating that none of them are assigned yet, although the frequencies are assigned.
Now you need to transfer frequency assignments to peak assignments.
33. Automatically generating peak assignments from frequency assignments
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.
34. 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.
35. 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.
You now should see output in the text 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 dN(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 Å). 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 text 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.
36. Reading in the database containing fully-assigned patterns
Now select the buildup experiment using the Experiments table, highlighting the fourth row and clicking the Draw icon.
The spectrum-specific shifts for this experiment are not exactly set yet, you need to adjust them in the next step:
The next step is to assign all the peaks with the help of these newly defined spectrum-specific chemical shifts.
37. Assigning the buildup automatically
After one or two minutes the auto-assignment is done. You should see something like this in the text 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.
38. Defining the scalar peak Find Peak By Number
Select the Peaks/Find menu item. Set these values:
Action Zoom+Color
Peak ID 1153
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. Peak Name D1 1:ASP-_13:HB1
Select the Measure/Scalar/Normalize menu item. In the control panel, set the Add One option and select OK.
In the next control panel, set the values:
Peak Name D2 1:ASP-_13:HB2
Distance 1.75
Now set the intensity plot (if you were in contour plot) and draw a full plot (View/Limits/Full Limits).
39. Defining the restraints Strong -1.0 2.5
Select the Measure/DISCOVER Restraints menu item. In the first control panel, set Restraint Class to NOE Distance and Action to Define, then select OK.
In the third control panel, set these values:
Medium -1.0 3.5
Weak -1.0 6.0
In the fourth control panel, leave Lower Bound at -1.0 and Upper Bound at 6.0 for the overlapped peaks. Select OK.
Yellow footprints appear on the plot, indicating the peaks from which restraints are generated. At the end of the procedure a message appears:
458 NOE distance restraints calculated
From these 42 were qualitative distance restraints because of partial overlap
16 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.
40. Writing the database
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.
41. Redefining the coordinates
Select the Assign/Read Coordinates menu item. From the list select znrddg.car and select OK.
This replaces the linear-chain coordinates of Zn-rubredoxin with the first DG-II structure coordinates and may take up to a minute.
42. Rerunning autoassignment for the buildup
First unassign the peaks by selecting Assign/Peak Assign/Unassign Peaks and leave the peak entity as xpk:buildup. Select 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.
43. Regenerating the restraints Strong -1.0 2.5
Select the Measure/DISCOVER Restraints menu item. In the first control panel, set Restraint Class to NOE Distance and Action to Define. Select OK.
In the third control panel, set these values:
Medium -1.0 3.5
Weak -1.0 6.0
And in the fourth control panel, leave Lower Bound at -1.0 and Upper Bound at 6.0 for the overlapped peaks. Select 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:
596 NOE distance restraints calculated
From these 46 were qualitiative distance restraints because of partial overlap
37 peaks were discarded because of assignment problems
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.
Select OK.
In the second control panel, enter the Filename as zn_viol01.txt and leave the other parameters at their defaults. Select 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 Å was violated in 14 conformations out of a total of 20, and the violation average was 0.17 Å. The average distance measured in the 20 conformation is 4.64 Å and the calculated distance based on ISPA is 2.78 Å. 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 text 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. Select 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.
45. 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.
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 with this classification to the disk. This file can be imported into Insight II and can (for example) be rendered on the ZNRDDG molecule.
46. Writing the database
This action writes out the znrddg.pks, znrddg.ppm, znrddg.asn, and znrddg.rstrnt files.
47. Exiting Felix
To exit Felix, select the File/Exit menu item.
The topics covered in this lesson are:
1. Setting up for the lesson
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).
Select the Assign/Project menu item.
3. Setting up the database Selection mcp1_lec.car
In the second control panel, set this value:
This procedure typically takes several seconds.
After this step is successfully completed, a library should be defined. The library is an ASCII file, as described under Assign in the 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 select OK.
In the fourth control panel, select pd.rdb and select 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).
Select OK.
Select 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.
The program plots a density or contour plot of the 15N-HSQC spectrum using the parameters you defined.
Now another control panel appears.
The spectrum-specific tolerances are important to define and are used in many automated and semi-automated procedures.
5. Adding the 15N-HMQC-TOCSY experiment to the projects
Again select the Assign/Experiment menu item.
This brings up the Experiments table.
Select 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 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.
6. Repeating Step 4 for the 15N-HMQC-NOE spectrum
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.
This command reads in the peaks and displays them in a spreadsheet. The peaks are also displayed as boxes.
Select the second row in the Experiments table and click the Draw icon to display the tocsy spectrum.
Select the File/Import/Peaks menu item. Set the Selection parameter to mcpn15tocsy.xpk and select OK.
When the program asks you whether to overwrite the entity, select OK.
Select the third row in the Experiments table and click the Draw icon to display the noe spectrum.
Select the File/Import/Peaks menu item. Set the Selection parameter to mcpn15noe.xpk and select OK.
When the program asks you whether to overwrite the entity, select OK.
Now you have a full peak set defined for both experiments.
8. Selecting the HSQC spectrum
Select the HSQC spectrum in the Experiments table. Press <Ctrl>-f to obtain the full plot.
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
You see a control panel with several options. The program tries to automatically fill in reasonable values
.
Information about the current stage of prototype pattern collection appears in the text 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 text window:
Nr of protos generated : ( 52)
The 3D protopattern detection took 49 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
You get a third control panel with several options. The program tries to automatically fill in reasonable values.
Information about the current stage of prototype pattern collection appears in the text window. After one minute, the prototype pattern collection is finished, and the following information appears in the text window:
Nr of protos generated : ( 13)
The 3D protopattern detection took 61 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
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
For the following action, you need to have room for two spectral frames. D1 D1 Define Jump on
Select the Preference/Frame Layout command and click the Add New button. Select Tile as the Rearrange Layout parameter.
For the current frame (Frame 2), select the hsqc experiment in the Experiments table.
Select the Preference/Frame Connection menu item. In the control panel, set First Frame to 2 and Second Frame to 1. Select Custom and select OK.
In the second control panel, set these values:
D2 Null
D2 D3
Null Null
After you finish with the control panels, the two spectra are connected. 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
Select the hsqc experiment and draw a full plot, as in Step 8.
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 select 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.
After several seconds, 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. Zoom in on that peak (View/Limits//Set Limits), then type. and move the subsequent 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.
Select the Assign/Frequency Clipboard/Zero Clipboard menu item to clear the clipboard.
Now you are ready to add frequencies to this clipboard.
You can check what is in the clipboard by listing it (Assign/Frequency Clipboard/ View Clipboard), and the result is printed in the text 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.
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. Select OK.
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
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. Type pa66 for the Pattern parameter, and leave the Number of freq as 4 and the Mode as New. Select OK.
A new pattern with name pa66 is added:
No such pattern pa66, adding it!
16. Scoring the patterns Min Atoms N
Select the Assign/Residue Type/Score Residue Type menu item. Set these values:
Max Std Dev 3
Scoring Method All Atoms
Patterns All
Database Store
Use COSY Peaks None
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), H,i-HN,i+1(-Ni+1), and H,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 select All. Set the tolerances to 0.02, 0.04 and 0.1. Select 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. Select OK.
Now select the Assign/Spin System/Auto Root menu item, set Patterns to All, and select OK.
18. Performing neighbor searches
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
------------------------------------------