Instructions

>>> top.activateAriaSetup()

The resulting panel allows us to control which data goes to the ARIA calculation from the CCPN project. For more fine-grained control you need to use the ARIA GUI; for example to change the annealing protocol.

Create a new job for ARIA to work on by pressing the green "New Run" button. This "Run" object links together all the data that goes into the same calculation. Ensuring that the {Input Data} and {Peak Lists} tabs are selected (they ought to be by default), change the pulldown menu at the bottom right to N-NOESY:182:1 and click [Add Peak List]; this will state that in this run ARIA should use this data. Now swap to the {Restraint Lists} tab and add restraint lists in a similar way: Select the list name from the pulldown menu and click [Add Restraint List]. Do this for all of the restraint lists, i.e. the H-bond, two distance and dihedral restraint lists. Now that all of the input data is set we have to tell ARIA how to run on the data.

Move to the {Run Settings} tab, found at the top. Here you can see some of the settings to control the ARIA run. In the lower table make sure that the "Ambiguous protocol?" column set to "Yes" for the "Ambig" distance restraints and "No" for the "Unambig" distance restraints. If we wanted to run the ARIA structure calculation locally (noting you will have to have a CNS executable and ARIA itself available) we can [Launch ARIA GUI], make relevant changes, save the ARIA project and then [Setup Project], which will put the ARIA data in a state that it can be run from the command line in the usual way.

For this demonstration you may run the ARIA calculation remotely using the prototype CCPN Grid service. To do this simply press the [Submit to CcpnGrid] button. For this demonstration use the user identification ''test" and the password "test123". We will not wait for the final structure calculation, which will take some time (although this protein will take less than an hour on an unloaded server), but instead we will look at a structure calculation that has already been completed, and which uses exactly the same data as you have been looking at in the CCPN project.

Via a web browser go to the page http://webapps.ccpn.ac.uk/ccpngrid/status: if you do start a grid calculation this is the page that you get to via the [Show web page] button. If asked, log on with UID: ''test" password: "test123" . Here you will see various ARIA calculations, one of which will have status "Finished" and be entitled ''CcpnStructCourse...". Click on the [Results] button for this job and look at the available data. Note  that we have the option to download an updated CCPN project which contains the newly calculated structures and a violation analysis, all of which remained linked to the NMR data in the CCPN project; so that for example we can easily jump from a violation to the offending point in the spectrum.

Importing Structure Calculation Results

You can see the CCPN project that resulted from this completed ARIA run by loading the project CcpnStructCourse3c: In the menu bar select M:Project:Open Project then [Yes] to close the current project and [No] to not save. Navigate to find and select the CcpnStructCourse3c project, then click [Open]. Although we are using a pre-prepared CCPN project, to use the CCPN project that was generated by ARIA on the CCPN Grid it simply has to be downloaded. We will now have a look at the result of the structure calculation by using Analysis.

Structures

In the data that comes back from ARIA we will see that two structure ensembles have been entered into the CCPN project; one from the last ARIA iteration and one after the water refinement stage. To see the structures go to M:Structure:Structures. If you go to the {Structure Models} tab select the [Superpose & Calculate RMSDs] button at the top right and then [OK] to see how well the structural models align. To see the structure in a 3-dimensional representation for whichever is selected in the table click the [Viewer] at the top.

The controls for the structural viewer are as follows:

• Rotate with middle-click & drag.

• Zoom with the mouse wheel, or middle-click, <Shift> & drag.

• Move with middle-click, <Ctrl> & drag.

The mouse right-click brings up a menu that allows you to change the display mode, spin, and print the structure. The left-click is used for atom selection. Try the atom selection by first ensuring that the N-NOESY peak list is selected at the top, click on an amide location on the structure (i.e. a blue atom) and then click [Show Peaks]. This will show a table containing all of the peaks that relate to connections from the selected atom in the structure. The numbers on the dashed lines represent the distances between the atoms.

The same sort of functionality is present in the Edit Assignment popup (M:Assignment:Assignment Panel). - If you look at the NOESY spectrum in window2, and assign a peak (by pressing <a> with the cursor over a peak), you can see assignment possibilities via the [Show On Structure] button. Also note that because we now have a structure the Edit Assignment popup will show distances between one peak assigned 1H resonance and the 1H possibilities in another peak dimension.

Referring back to the restraints popup (M:Structure:Restraints & Violations {Restraints}), set the structure pulldown menu to "2"; the structure from the ARIA/CNS water refinement. Next set the restraint list as one of the Distance lists. You will see that the "Struc Value" is now filled-in for the restraints and you are are able to select any restraint rows (using <Shift>/<Ctrl> keys) and then click [Show Selected On Structure] to illustrate graphically where on the loaded structure the restraints apply.

Restraints & Violations

In the Restraints & Violations popup (M:Structure:Restraints & Violations), choose Restraint Set "2" at the top; these are the results from the ARIA run (set 1 was the input) and choose the {Restraint Lists} tab. You will see that ARIA has generated one or more restraint list for each peak list and restraint list that was used as input data. Choose the second row labelled "ARIA_REJECT_run1_NOEs_it8", which represents the peaks/restraints that ARIA rejected, and select the {Restraints} tab. From the Structure pulldown menu at the top check that "2" is selected (the water refined ensemble) and in the Violation List pulldown menu table select "1"; you will hopefully see that many of the rows become orange/red, indicating that these restraints caused violations. If you change the Restraint List pulldown menu to a list that is not labelled as "REJECT" you will see that there are far fewer violations.

Choose a restraint row for one of the violated (red/orange) restraints and click [Show Peaks]. This will open a table containing the peak (or peaks) that gave rise to the restraint. Note that a restraint may be linked to more than one peak, for example where there are symmetry related peaks that correspond to the same close resonance pair. In this Selected Peaks table click on the peak row and then the [Find Peak] button at the top. You will be whisked to the point in the spectra where the peak resides. From here you may choose to look at the peak assignment by using the <a> key with the mouse over the peak. You can get to the same point directly from the restraint by using the [Assign Peak] button from below the Restraints table. Note that ARIA has generated an assigned peak list, so when we look at the N-NOESY spectrum we see two, differently assigned, peak lists on top of one another. To switch off the new peak list in the spectrum window you can click on the [Peaks] button at the top and toggle the [N-NOESY:182:2] option.

Now go back to the list of restraints. Click on the heading of the "Mean Viol" column then scroll to the bottom of the table to see the largest violation. Select the last red row (probably restraint number 77) and click [Assign Peak]. In the Assignment Panel that appears/updates you will see that the original peak that was used to make the restraint does not have an assignment an the second dimension. This peak was not satisfactorily assigned during the structure calculation process, but this is not surprising given that no care was taken in generating an initial set of seed assignments; we deliberately wanted a project with lots of interesting violations. For the second "F2" peak dimension in the Assignment Panel click on the "Dist" heading (mid-right table) so that the possible assignments are presented in order of distance from the assigned amide proton. Hopefully, you will see that there are two resonances "3GluHga" and "3GluHgb", from the same residue as the amide, at 2.7 Angstroms and that the chemical shift difference to the peak position "Delta" is 0.044 PPM. These options are close enough in both chemical shift and structural distance to be good assignment candidates, thus click on both the "3GluHga" and "3GluHgb" rows for the second dimension to assign them. The peak will now have contributions to both resonances.

Repeat this operation once more for the second to last restraint (number 22), again clicking [Assign Peak]. This time you will see that there is an assignment option for "36IleHa" which seems plausible. Although this is not the closest possibility in the structure ensemble (at least the what we have so far) it is chemical shift difference is fairly small, unlike the "41ProHdb" option. Once we have checked all of the rejected and seriously violated restraints, adjusting their peaks accordingly we would go through another round of structure calculation, with a better starting set of NOE assignments. Although we can go though each problematic restraint in a fairly manual matter, there is a more automated procedure for checking NOE peak lists and curating their assignments using chemical shift and structure information.

Using Structures to Assign NOE Contributions

An alternative, more detailed, method of looking at NOE assignments is via the M:Assignment:NOE Contributions option. This is designed to make the whole process of comparing NOE peaks to a structural model efficient. Select this and in the "Peak List & Display Settings" choose the "NOE Peak list" for the NOESY experiment you are currently looking at, then double click the "Use?" column in the lower table for the relevant window, so that it becomes "Yes". Also set ''Structure Display" to "Assigned" and 'Mark Peak' to true. Now move to the {Peak Assignments} tab, select the peak in the spectrum window (left click & drag) and in the NOE Contribution popup click [Selected Peak]. This will show you, in terms of the close chemical shifts and distances from the selected structure, what the likely NOE assignments are.

By clicking on the rows in the NOE peaks table you will see that several things happen automatically: The view of the selected windows (in this case the selected window2) zooms and marks the selected peak; the graphical structure view highlights the assigned atom connections from the peak; and the lower table of the NOE Contributions popup shows the structurally possible, shift-matched resonance pairs ordered in terms of shift and geometric distance. Select the first peak in the table (10Tyr) and you will see that the top row for the possible assignments is "9SerHbb", which is close in chemical shift and only 2.939 Angstroms away. Click on the "9SerHbb" for the second column in the table and then [Assign Selected]. The will see that the upper table, spectrum window and structure view all update to reflect the new assignment.

Next, in the upper table select peak number 4 (62Glu) and you will see that the assignment possibilities are not so clear cut. Here, selecting [Predict Peaks] will use the entered structure and the known chemical shift values to predict the positions of peaks, near to the selected real peak, which correspond to close resonance pairs. These artificial peaks are labelled with the structural distance and are contained in an entirely separate list to the real peaks (so nothing is contaminated and they can be removed easily). In this instance it is probably appropriate to assign the peak to bo

Assignment & Structure Validation

th of the top assignment possibilities (from residues 62Glu and 31Ala). While holding down the <Ctrl> key select both the "63AlaH" and "32LeuH" options in the table and click [Assign Selected]. We can confirm that this double assignment was the correct thing to do by looking for "return" peaks that show the reciprocal connection (looking from the point of view of the other amide). Clicking on [Find Reciprocal Peaks] you will see that the spectrum window splits into separate panels, with the original amide location on the left. Strips 2-4 show other amide positions with peaks that may possibly reciprocate the same proton-proton connection. Peaks for 63AlaH and 32Leu are indeed present and confirm the assignment in the second dimension we just made. Note also that 36Leu shows a potential return peak, but this is too far away in the structure and so does not appear in the table.

Note that if we change peak assignments then we have the choice of making (or letting ARIA make) a new set of restraints. Alternatively we can curate the existing restraints using the [Update Assignment From Peak] button (see M:Structure:Restraints & Violations {Restraints}), which will alter the distance restraint to reflect this new assignment.

Assignment & Structure Validation

The final part of these tutorial exercises is to look at how we can use the CING software to analyse and validate NMR and structure data. Also, we will consider the data checks that can be performed within Analysis, without the need for external programs, for example to find chemical shift outliers and bogus assignments..

Structure Validation

In the main menu select "M:Structure:CING: Validate Structures" and click the green [New Run] button in the upper right corner. This run specification will contain all of the data that CING will analyse. When a new run is made the {Structures} table will fill with the various models from an ensemble. Accepting this, we move on to the {Shifts & Measurements} tab. Here click [Add Measurement List] to add the shift list to the CING analysis. Move to the {Peak Lists} tab, and add the N-NOESY:182:2 peak list (this is the one that ARIA made) by selecting it from the lower right pulldown menu and selecting [Add Peak List]. Finally in the restraint lists tab add restraint list "Distance-2:1". This restraint list is the one that came back from ARIA and consequently lives in the second restraint set. Note that in general you may select as much data as you like, but we are just selecting a smaller subset for demonstration purposes.

Select the {Run Settings} tab. To submit the analysis press the [Submit Project] button. Note that this may take some time, so you may wish to let the demonstrator make a real submission and view some previously calculated results, thus sparing the iCING server. Although, feel free to use this server generally.

To view some precalculated validation data look at the following URL in a web browser:

http://nmr.cmbi.ru.nl/CASD-NMR-CING/data/GR/CGR26ACheshire/CGR26ACheshire.cittp

Assignment Quality Control

At the menu option M:Assignment:Quality Reports you can access a suite of analyses which generate automated reports about the quality and completeness of your assignments. The first tab simply lists how many of each kind of residue and atom type you have resonance assignments for. The second tab shows statistics for how many NOE (and other through-space) connections you have across your molecular system.

While the first two tabs are dedicated to analysing completeness, the last two tabs are dedicated to finding mistakes and inconsistencies in assignments. These tables look at the same kind of issues, but one presents the information from a resonance point of view, and the other from a peak point of view. Most notably the assignments are checked for the following criteria:

• An assignment to a given atom is duplicated.

• The standard deviation for a chemical shift is large.

• If a peak position is far from the chemical shift average (irrespective of a shift's deviation).

• A resonance has an impossible number of covalently bound partners: e.g. an amide proton assigned to two HSQC peaks cannot be linked to both nitrogen resonances.

• The chemical shift for an atom assignment is unlikely given the known chemical shift distributions (BMRB).

• If peaks have unusual intensities or sign.

When something is potentially wrong, then cells of the tables are coloured red. A highlighted cell doesn't mean that something is definitely wrong, just that it warrants an explanation or further investigation. To find the peaks responsible for any aberration, simply click on the row in the table and click [Show Peaks]. In the resulting table you can assign or find any of the peaks in the various spectrum windows.