Making Dihedral Angle and Distance Restraints

The penultimate part of the exercise is to look at how we can use a CCPN project to get peaks lists and restraints (both dihedral and distance) that can be used in an ARIA structure calculation, and how we can pass intermediate structural information back into Analysis to help with violation analysis and NOE peak assignment.

Open an existing project

For this last section we will be using programs that are part of the Extend-NMR software collection, so rather than starting Analysis we will initially launch the Extend-NMR graphical interface on the command line by typing:

-> extendNmr

When the Extend-NMR menu bar has appeared:

, select M: Project: Open Project. Navigate to find and select the CcpnCourse3a project, then click [Open].

You might get a warning that various files have moved location. You might also get a dialog with a list of spectra paths (because those also have moved location).  If the paths are all in grey then just click the [All Done!] button at the bottom. If any path is in red then Analysis cannot find the corresponding spectrum data file, so either you need to tell Analysis where it is (by double clicking the path cell and navigating to the correct location) or accept that that particular spectrum will not have its contours displayed. When the project data is loaded select M: CcpNmr: Analysis and a blue NOESY spectrum will hopefully appear.

Making Restraints From Assigned Peaks

To make a list of distance restraints from the assigned peaks in an NOE peak list first go to M: Structure: Make Distance Restraints. At the top of the popup, change the peak list to C-NOESY:173:1 and set the Restraint Set pulldown to "1" . We can leave all of the other parameters alone for demonstration purposes. The Restraint Distance Params section would allow us to specify how the NOE peak intensities relate to the distance bounds of any generated distance restraints. The default method is to calculate a target distance as peak height raised to the power of -1/6 multiplied by some scaling factor, such that the reference intensity (in this case defaults to the peak list's average volume) exactly corresponds to the reference distance (in this case 3.2 Angstroms). The upper and lower bounds of the distance restraint are calculated as fractional changes from the calculated target distance (default is 20% above and below) while observing absolute minimum and maximum values for the bounds (1.72 & 8.00 Angstroms respectively by default). The {Residue Ranges} and {Chem Shift Ranges} tabs would allow you to make only restraints for specific assigned regions of your molecule or for specific shift ranges.

To calculate restraints for assigned peaks from the selected peak list simply press [Make Assigned Restraints]. After a short pause you will see the Structure: Restraints and Violations popup appear. This shows that you have one restraint set (a way of grouping related restraints and violations) containing a H-bond restraint list, which was already loaded via the FormatConverter. Click on the row of the restraint list in the central table and then click on the {Restraints} tab. Note that you can also get to this point via the M: Structure: Restraints and Violations option.

The restraints popup will appear and in its table you will see the restraints listed, mostly as green coloured rows. Note some restraints also have following grey rows. These grey rows indicate restraints that are ambiguous, i.e. a possible connection between two different pairs of 1H resonances. Note that such ambiguous restraints can represent logical uncertainty (before an NOE is resolved) or real physical ambiguity where a peak is caused by two or more overlapping pairs of resonances.

Making Restraints From Unassigned Peaks

There is a second common way to generate distance restraints, which is to match the chemical shifts of resonances to NOE peak positions, thus generating potentially highly ambiguous distance restraints. Such restraints would typically be filtered to select only the correct contributing resonance pairs, by iterative structure generation and violation analysis in a program like ARIA. Firstly, we could leave the matching of chemical shifts to the ARIA program by handing the program peak lists rather than restraint lists, which is what we we will demonstrate for the N-NOESY data. However, it is also possible to make such restraints in CCPN. Accordingly, the {Shift Match Tolerances} and {Network Anchoring} tabs in the Structure: Make Distance Restraints popup allow you to generate such distance restraints for peaks which do not have assignments. To generate distance restraints by shift matching, firstly set the peak list to "C-NOESY:173:1" and then click [Make Shift Match Restraints]. This command uses the current settings, but {Chem Shift Ranges} and {Shift Match Tolerances} are only relevant for this command.

In the case of the shift-matching method potentially ambiguous distance restraints are generated by simply matching peak positions to close chemical shifts. In the case of network anchoring method, chemical shifts are also matched to peaks, but the ambiguous possibilities are refined by selecting only NOE assignments from amongst the possibilities that are supported by other, assigned NOEs or covalent structure. Say, for example, that a peak could arise from a number of resonance pairs. Two resonances A & B are more likely to be a correct assignment for the peak if we know that they are close to (or bound to) the same intermediary resonance, C and therefore must be close to each other.

Merging and Splitting Restraint Lists

To prepare these newly generated restraint lists for the ARIA calculation we will merge and split them in order to generate restraints that are separated into "Unambiguous"  and "Ambiguous" categories. In ARIA we do this so that the "Ambiguous" restraints and peaks follow a different protocol; they enter the calculation after the unambiguous, more certain, restraints have formed the initial structure.

Via M: Structure: Restraints and Violations, {Restraint Lists}, merge the two lists that derive from the C-NOESY experiment by clicking on the two relevant rows (probably numbers 2 & 3) while holding down the 'Ctrl' key. Now click [Merge Lists] at the bottom and [OK]. You will see that the restraints have been combined and there is now only one list from the C-NOESY:

Then for the remaining, enlarged restraint list, select its row and click [Split Ambig/Unambig]. These are now ready for input to ARIA.

Dihedral Restraints

Next we will generate restraints in a different manner; dihedral restraints from backbone chemical shifts. We will be using a program called DANGLE (Dihedral ANgles from Global Likelihood Estimates) which is embedded within Analysis. DANGLE estimates dihedral angles from chemical shifts in a similar manner to TALOS; i.e. it matches a chemical shift & sequence query to a structural database of known PHI/PSI angles and chemical shifts. However, DANGLE uses a different (Bayesian) method to produce an angle estimate and tolerance, compared to TALOS. The idea is to use Bayesian inference to infer what the range of likely PHI/PSI angles might be (using the chemical shifts) by checking all PHI/PSI combinations in 10 degree square bins to see how well such angles can be used to explain the data. Such an analysis allows for the user to see uncertainties in the angle predictions, including where the chemical shift to structure mapping is redundant and there are multiple regions in the Ramachandran plot which could explain the chemical shift data.

To run DANGLE select M: Structure: DANGLE: Predict Dihedrals. Note at the top that the Chain should be set as "GI:A", the Shift List as "ShiftList 2:2" and Max No. of Islands as 2. This simply specifies which data to use and how strict the analysis should be. Using two islands means that we will reject predictions that result in more than two discrete regions of the Ramachandran plot. To start the analysis press [Run Prediction] and accept "Run1" as the name for the job by pressing [OK] at the opportune moment. Please be aware that DANGLE will take several minutes to finish the calculation.

Once the calculation is over you will see the main table filled in with PHI and PSI backbone dihedral angle predictions and their associated error ranges. Further, if you select a row in the main table you will see a plot in Ramachandran (PHI/PSI) space of where the likely angles are deemed to be. Click on the "7 Ser" row and note that there is a lot of red colour in the chart, indicating that DANGLE was not able to make a distinct choice of PHI/PHI: you should not use such a prediction in a structure calculation. Click on the [Next] button to get to "8 Lys". The prediction for this residue is somewhat better, and you could use this in a structure calculation (it has one discrete region) although the error bounds for such a dihedral restraint would be suitably large. Click on the  "12 Glu" row. This residue has a very precise range of predicted PHI/PSI angles. Such a residue could be used in a structure calculation with a high degree of confidence and proportionately narrow error margins:

Note that DANGLE also predicts the secondary structure of the residues, but that this calculation is not made from the angles, but directly from the measured secondary structures in the shift-structure database. To make the restraints themselves set the Restraint Set to "1", which will place the PHI/PHI dihedral restraints with our existing distance restraints and press [Commit Constraints]. View the generated restraints by going to M: Structure: Restraints & Violations, {Restraints}. Note that if you have a structural model for your protein you can see how the model's angles match with the DANGLE prediction.

Back to Course Day 3