NOE assignment & protein structure calculation using ARIA

Structure calculation

If for any reason you are not confident about the state of your CCPN project at this stage of the demonstration before we do a structure calculation you may like to open a pre-prepared CCPN project, which has all of the expected restraints present: In the Extend-NMR 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 CcpnCourse3b project, then click [Open].

To start the structure calculation using the restraints we have setup, return to the Extend-NMR GUI and select the {ARIA 2} tab. This 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 just below the ARIA logo. 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 peak list and the "Ambig" distance restraints and "No" for the "Unambig" distance restraints. If we wanted to run the ARIA structure calculation locally (notice you will have to have a CNS executable 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 we are using the user identification ''test" and the password "test123", which is passed to the CCPN server automatically for the demonstration. 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.

Either click on [Show web page] in the CCPN Grid progress popup that appears:

or using a web browser go to the page http://webapps.ccpn.ac.uk/ccpngrid/status. 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 ''CcpnCourse3b...". Click on the [Results] button for this job and look at the available data, making note of the fact  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:

You can see the CCPN project that resulted from this completed ARIA run by loading the project CcpnCourse3c: In the Extend-NMR 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 CcpnCourse3c project, then click [Open]. Select the {ARIA 2} tab and then the {Output Data} tab contained therein, you will see a list of all of the data that ARIA has passed back to the CCPN project; restraints, structures & peaks. We will now have a look at this data by using Analysis. If you do not have Analysis open, open it by going to M: CcpNmr: 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 an ensemble (G1:1 or G1:2) and click [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 Assignment: Assignment Panel 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 Assignment: Assignment Panel popup will show distances between one peak assigned 1H resonance and the 1H possibilities in another peak dimension (here it shows a distance of 4.052 Å between 62GluHga and 60GlyH):

Referring back to the Restraints (M: Structure: Restraints and Violations {Restraints}), set the structure pulldown menu to "2", the structure from the ARIA/CNS water refinement, and 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.

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