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:
The following files from the $BIOSYM/tutorial/felix directory are required for this lesson. Please copy these files to your working directory:
zh.mat
zc.mat
zn.mat
zb.mat
znrdlec.car
znrdlec.mdf
dqf.xpk
tocsy.xpk
noe.xpk
noe450.xpk
noe450.vol
zn_model.dba
zn_viol01.txt
znrddg.car
znrddg.mdf
In your working directory, enter felix at the system prompt to start the program. If you get the RESTORE LAST SESSION dialog box, select CANCEL.
Select File/Open, and set the File Type to DBA. In the Files list select zn.dba and select OK to build a new database.
When FELIX informs you that no project was found select OK, and 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 Selection to znrdlec.car.
This procedure typically takes several seconds. Then the program asks for the library. The library is an ASCII file, as described in the Assign section of Chapter 1, Theory, in the FELIX User Guide. FELIX contains a standard library for proteins and DNA (pd.rdb) which you should read in.
This is the protein/DNA library. A few seconds later the project setup procedure finishes.
5. Viewing the project entity through a spreadsheet
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.
6. Adding an experiment to the projects
Select the Assign/Experiment menu item to define new experiments in the assignment database. When the control panel appears with the names of matrices in the ./ directory, select zc.mat (the DQF-COSY spectrum).
Set the following parameter values for the plot using the 2D Display Parameters control panel:
Leave the other parameters at their default values and select Apply.
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.
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:
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
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:
Leave the remaining parameters at their default values and select OK.
In the next control panel, set these parameter values for TOCSY:
In the next control panel set these values:
Next set these parameter values:
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.
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 on page -200 in Chapter 4, Processing, visualization, and analysis interface (1D/2D/ND), 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).
Select the File/Import/Peaks menu item. Set the Filter parameter to ./*.xpk and click Filter. Set the Selection to dqf.xpk by clicking the filename in the Files list box. Leave FELIX Peak Table Name at its current setting (xpk:cosy), since your current experiment is a DQF-COSY.
Select the File/Import/Peaks menu item. Set the Filter parameter to ./*.xpk and click Filter. Set Selection to tocsy.xpk by clicking the filename in the list box. Set FELIX Peak Table Name to xpk:tocsy, and select OK.
When the query box appears, asking about overwriting the entity, select OK.
Next you repeat the procedure for the NOE spectrum.
Select the File/Import/Peaks menu item. Set the Filter parameter to ./*.xpk and select Filter. Set Selection to noe.xpk by clicking the filename in the list box. Leave FELIX Peak Table Name as xpk:noe and select OK.
When the query box appears, asking about overwriting the entity, enter OK.
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.
Set these parameter values in the control panel:
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 using the More... button instead of the OK button. In the next control panel leave all parameters at the default values:
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.
In the remaining part of the control panel, set these parameter values:
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.
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. Select 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.
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.
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.
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:
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 fre- quencies:
# 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.
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:
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
Select the Assign/Residue Type/Score Residue Type menu item. 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, Database to Store, and Patterns to Specific. Select OK.
In the text window, a report is generated of the probabilities that this pattern belongs to a certain type of amino acid residue:
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:
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.
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 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.
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 in the pattern. Now click 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 value 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. Select Yes.
Now repeat the procedure for the other frequencies in this pattern. When you are finished, select Quit.
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.
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!
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
The results should be similar to these:
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
For the next prototype pattern select 55th. Clear the frequency clipboard (Assign/Frequency Clipboard/Zero Clipboard) and then select the Assign/Frequency Clipboard/Copy Proto to Clipboard menu item and copy prototype pattern 55. Display the strip plot on the TOCSY experiment using the Assign/Frequency Clipboard/Strip Plot Clipboard menu item.
You can see that, in the column of 9.194 ppm, there is an extra frequency
around 2.5 ppm.
Zoom to the 2.5 ppm peak (select View/Limits/Set Limits or use the Zoom icon).
Add this frequency to the frequency clipboard by selecting the Assign/Frequency Clipboard/Add One menu item and setting the Frequency parameter to W1 2.664909 and the Nucleus 1 to HX. Select OK.
Score the pattern as in Step 20. The result is:
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:
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:
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.
The result should be similar to:
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:
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:
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.
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
Residue type of pattern pa4 is set to ile
The probability for pa4 to be ILE is : 1.000
After matching the pattern against the ILE residue, you can assign the frequencies.
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:
27. 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, set the Pattern parameter to pa5 and leave 4 for Number of frequencies and New for the Mode parameter. Select OK.
The residue type scoring appears in the text window as:
with the most likely candidates as phenylalanine and cysteine. Match against these two residues:
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.
Select the Assign/Neighbor/Find Neighbor Via 2D NOE menu item. Set these values:
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 select OK.
After one or two seconds the results are printed and stored:
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).
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).
You now should see the results as a tile plot of the inter-pattern peaks.
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.
30. 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:
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
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.)
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.
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:
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
Select the Assign/Peak Assign/Autoassign Peaks menu item and set these values:
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
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).
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.
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:
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:
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
Select the File/Open menu item. Select the zn_model.dba file from the list and select DBA for the File Type parameter. Select OK.
Close the Spinsystems and Experiments tables and reopen them using the Edit/Spin Systems and Assign/Experiment menu items, respectively.
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:
Select the Assign/Spin System/Auto Update Specific Shifts menu item. Set the Spectrum Specific Shift parameter to the buildup experiment and Patterns to All. The D1 and D2 Tolerance should be set to 0.015. Select OK.
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
Select the Assign/Peak Assign/Autoassign Peaks menu item and be sure that these values are set:
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.
Select the Peaks/Find menu item. Set these values:
List this peak with the Assign/List Peak menu item:
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:
Now set the intensity plot (if you were in contour plot) and draw a full plot (View/Limits/Full Limits).
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.
Set these values in the next control panel:
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:
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.
Select the File/Export/Restraints menu item. Set Peaks and Resonances and Restraints to on.
For the filenames enter: (Peak Intensity File, Chemical Shift File, Assignment File, Restraint File) znrdlec and set the Type to DISCOVER.
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
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.
Select the Assign/Peak Assign/Autoassign Peaks menu item and set these values:
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
Select the Measure/DISCOVER Restraints menu item. In the first control panel, set Restraint Class to NOE Distance and Action to Define. Select OK.
Set these values in the second control panel:
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:
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.
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.
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.
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:
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.
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.
Since this is homonuclear data, select the Assign/Chemical Shift Index/HA Chemical Shift Index menu item. Set these values:
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.
Select the File/Export/Restraints menu item. Set Peaks and Resonances and Restraints to on.
For the filenames enter (Peak Intensity File, Chemical Shift File, Assignment File, Restraint File) znrddg, set the Type to DISCOVER, and select OK.
This action writes out the znrddg.pks, znrddg.ppm, znrddg.asn, and znrddg.rstrnt files.
The topics covered in this lesson are:
The following files from the $BIOSYM/tutorial/felix directory are required for this lesson. Please copy these files to your working directory:
hsqc.mat
mcpn15tocsy.mat
mcpn15noe.mat
mcp1_lec.car
mcp1_lec.mdf
hsqc.xpk
mcpn15tocsy.xpk
mcpn15noe.xpk
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).
In your working directory, enter felix at the system prompt to start the program. If you get the RESTORE LAST SESSION dialog box, select CANCEL.
Select File/Open, and set the File Type to DBA. From the Files list select mcp.dba and select OK to create a new empty database.
FELIX informs you that no project was found in the database. You have to acknowledge that you want to build a new project by clicking OK.
FELIX then asks you for a new project name. The default name in this new dialog box is asg:project. You can enter another name if you want (for example, mcp:project).
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 in the Assign section of Chapter 1, Theory, in the FELIX User Guide. FELIX contains a standard library (pd.rdb) which you should read in.
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).
Set these values for the plot, in the 2D Display Parameters control panel:
Leave the other parameters at their default values and select Apply.
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 Title should be descriptive, but not too long (for example, hsqc is appropriate for this spectrum). Set the parameters to these values:
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
This brings up the Experiments table.
Go to the Experiments table and 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).
Set these values for the plot using the 3D Display Parameters control panel:
Leave the other parameters at their default values and select Apply.
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.
The Title should be descriptive, but not too long (e.g., tocsy is appropriate for this spectrum). Set the parameters to these values:
6. Repeating Step 4 for the 15N-HMQC-NOE spectrum
Again select the Experiment/Add menu item in the Experiments table.
In the next control panel select these values:
Select the first row in the Experiment table by clicking it to chose the hsqc experiment. Then click the Draw icon.
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. Select OK.
When the program asks you whether to overwrite the entity, select OK.
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 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
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 select OK.
In the second control panel, set the Method to 3D HS(M)QC-TOCSY and select 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:
Now click More.... and set these 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
Select the Assign/Collect Prototype Patterns menu item. In the control panel, select the Double Resonance option and select OK.
In the second control panel, select the 2D HSQC + 3D HS(M)QC-TOCSY option and select 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:
Now click More... and set these values in the resulting control panel:
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
Select the tocsy experiment using the Experiment table: click the second row and then click the Select Experiment 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
For the following action, you need to have room for two spectral frames.
Select the Preference/Frame Layout command and click the Add New button. Select Tile as the Rearrange Layout parameter.
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.
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).
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.
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 select OK.
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.
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. Select 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 select OK.
In the second control panel, do not change any values, just select 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:
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!
Select the Assign/Residue Type/Score Residue Type menu item. Set these values:
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:
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.
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:
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.
18. Performing neighbor searches
Select the Assign/Neighbor/Find Neighbor Via 3D N-15 NOE menu item. Set these values:
Click More... and set these values:
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:
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. Select 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 text 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 Chapter 2, Tasks, in the 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:
Leave the other parameters at the default values and select OK.
After few seconds you will see a listing of several possible assignments, the beginning of which would look like:
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 text 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:
Leave the other parameters at the default value (1.0) and select OK.
The output in the text window should be similar to this:
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.
In the text 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.
Exiting FELIXThe topics covered in this lesson are:
The following files from the $BIOSYM/tutorial/felix directory are required for this lesson. Please copy these files to your working directory:
hncacb.mat
cbcaconh.mat
hnrnp.car
hnrnp.mdf
hncacb.xpk
cbcaconh.xpk
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).
In your working directory, enter felix at the system prompt to start the program. If you get the RESTORE LAST SESSION dialog box, select CANCEL.
Select File/Open, and set File Type to DBA. From the Files list specify hnrp.dba and select OK to create a new empty database.
Go to the menubar and select the Assign/Project item. When a dialog box appears informing you that no project was found in the database and asking if you want to build one, select OK.
In the control panel the default name is asg:project. You can enter another name if you want (e.g., rnp:project).
In the next control panel you need to select a molecule, in this case an extended chain of hnRNP C:
This procedure typically takes several seconds; a meter on the screen shows the progress.
After the setup is successfully completed, you should define a library.
The library is an ASCII file, as described in the Assign section of Chapter
1, Theory, in the 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).
Set these parameter values for the plot using the 3D DISPLAY PARAMETERS control panel:
Leave the other parameters at their default values and select Apply.
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.
Now another control panel appears.
The Title should be descriptive, but not too long (e.g., hncacb is appropriate for this spectrum). Set the parameters to these values:
It is important to define the spectrum-specific tolerances, since they are used in many automated and semi-automated procedures.
6. Repeating Step 5 for the CBCA(CO)NHN spectrum
In the table, select the Experiment/Add menu item.
Select cbcaconh.mat and set these values:
Choose cbcaconh for Experiment Title and set Type to 3D CBCA(CO)NH. Leave the Temperature at 298 and the pH at 7. Set Solvent to Water.
For D1, D2, and D3 Nucleus choose Carbon, Nitrogen, and Proton for the cbcaconh. For W1, W2, and W3 choose D3, D2, and D1, and for W1-W2 and W2-W3 Transfer, choose J-coupled. Set the Number of J Steps to 1. The spectrum-specific tolerances can remain at their defaults for cbcaconh.
In the Experiment table select the hncacb experiment (click the first row) and click the second icon (Draw). This draws the hncacb spectrum in the frame and also brings up the Real-Time Plane selection control panel, through which you can select new planes.
Then select the File/Import/Peaks menu item. Set the Selection parameter to hncacb.xpk and leave the FELIX Peak Table Name parameter at its current status (xpk:hncacb). Select OK.
When the query box asks you about overwriting the entity, click OK. This action reads in a peak entity and also displays it as a peak table.
Now select the cbcaconh experiment in the Experiments table and use the Draw icon to plot the last limits.
Select the File/Import/Peaks menu item. Set the Selection parameter to cbcaconh.xpk and select OK. In the new query box asking about overwriting the entity, click OK.
Now you have a full peak set defined for both experiments.
8. Selecting the HNCACB spectrum
Now reselect the hncacb experiment from the Experiments table. This time click the Select Experiment icon (first icon on the left).
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
Select the Assign/Collect Prototype Patterns menu item. In the control panel, select the Triple Resonance option and select OK.
In the following control panel, select the CBCANH + CBCA(CO)NH option.
Another control panel appears. The program tries to fill in its values automatically.
Set these parameter values in the third control panel:
After a couple of minutes, the prototype pattern collection is finished, and the following information appears in the text window:
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 first icon on the left (Zoom).
The region containing four peaks is displayed. Now connect the two spectra in which the prototype patterns were detected.
11. Connecting the HNCACB and CBCACONH spectra
For the following action you need to have room for two spectral frames.
Select the Preference/Frame Layout menu item. In the control panel, set the New Layout parameter to 2 Frames Up/Down. Select OK.
Now go the Frame 2 (clicking anywhere in its frame) and select the cbcaconh experiment through the Experiments table. Now return the first frame.
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 select OK.
After you finish with the control panel, the two spectra are connected. 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).
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 iconbar 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. Select 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 select 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. Select OK.
You can follow the progress in the text 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:
Select the Assign/Residue/Score Residue Type menu item. Set the Scoring Method to CACB only and select All for Patterns and Store for Database. Select 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:
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. Select OK.
After few seconds you will see many possible assignments; the beginning of the output will look like:
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) by clicking the maximize tool. Also make sure that the frame is in the foreground. 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 58, 41, 77, 20, 22, 2 and pa31. If the plot is too small, you can double the size of the frame by clicking the button in the left corner of this frame and selecting the Double item from the pulldown. (Note that this is possible only in regular mode - not in maximized mode.)
17. Setting the sequence-specific assignments for patterns 49, 64, 58, 41, 77, 20, 22, 2, 31, and 76
Go to the Stretches table and select the Stretch/Assign One Stretch menu item. Start the stretch at GLY_69 and leave the Assign Frequencies on. Select OK.
During execution the following lines appear in the text 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: