Structures
Loading Structures
Next we will load a protein structure, corresponding to the spectra that are present in this tutorial. This structure has been generated using the program ARIA, but in general such a structure could be from any source including early structure calculation runs or an homology model. To load the structure select M: Structure: Structures, {Ensembles}, set the molecular system as "MS1" then click on [Import] and select the file "G1.pdb" and finally [OK]. (If you don't already have the molecular sequence loaded at this point then say 'Yes' when asked if Phe and Tyr are to be considered fast-flipping on the NMR time scale.). To see the structure select it in the table and click [Viewer].
Referring back to the restraints popup (M: Structure: Restraints & Violations, {Restraints}),set the "Structure:" pulldown menu to "1", the structure we just loaded. You are now able to select any restraint rows (using 'Shift'/'Ctrl' keys) and then click [View Selected On Structure] to illustrate graphically where on the loaded structure the restraints apply.
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 selection.
The same sort of functionality is present in the Assignment: Assignment Panel popup (M: Assignment: Assignment Panel). - If you go back to the NOESY spectrum in Window 4, 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.
Making / checking NOESY assignments with structures
To make the whole process of comparing NOE peaks to a structural model efficient, Analysis has the M: Assignment: NOE Contributions option. Select this from the menu and in the {Peak List & Display Settings} tab of the resulting popup set the "NOE Peak List:" pulldown to "N-NOESY:182:1", set the Mark Peak toggle on and finally double click in the "Use?" column of the lower table over the "No" for window 4, so that it changes to "Yes":
Then go to the {Peak Assignments} tab. By clicking on the rows in |NOE peaks| you will see that several things happen automatically: The view of the selected windows (in this case the selected Window4) zooms and marks the selected peak; the graphical structure view highlights the possible atom connections that the peak could represent; and the lower table of the popup shows the structurally possible, shift-matched resonance pairs ordered in terms of shift and geometric distance. In the lower table clicking on an assignment row and [Assign Selected] sets that assignment for that peak. Also, 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. (e.g. row 12; 52PheH, 53AsnH, 52PheN see figure below)
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).
Calculating structural violations
Lastly, given the structure that we have loaded into Analysis and the distance restraints that we generated from the NOE peak list, we can investigate the compatibility between the structural model and the restraint distances to find violations.
Return to the Restraints and Violations popup (M: Structure: Restraints & Violations, {Restraints}) and select restraint list 1 from the pulldown menu and click [Calculate Violations]. After a short pause you will see that several of the restraint rows turn red:
These indicate that the restraint distances are inconsistent with (i.e. violate) the loaded structural model. The peak assignments from which this restraint list was generated obviously needs some work, so that hopefully a new and better structural model can be generated. Many of the violations are small, but some are large. The easiest way of zooming in on the large violations is to click twice on the |Mean Viol| column heading - this gives you the restraints sorted by violation, largest first. Taking for example restraint " 20LeuHba - 26SerH", the mean violation is 1.44 Angstrom. If you select this row and click [Assign Peak] you will see that an assignment in the F2 dimension, to 76LysHba, is probably missing: the chemical shift "delta" is small and the distance given the structure is close. You can click on the |Dist| column heading to get the assignment possibilities sorted by increasing distance. Clicking on the 76LysHba row in the Assignment: Assignment Panel popup will add 76LysHba as an ambiguous assignment:
Hopefully when the next set of restraints is generated (and maybe a new structure calculated) the peak will represent a shared contribution from two close 1H pairs, and the 26SerH - 20LeuHba restraint will no longer appear too short. Returning to the restraint table, with the extra resonance assigned to the peak and same restraint selected click [Update Assignment From Peak], which will alter the distance restraint to reflect this new assignment.
Previous: Restraints