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Pick and Assign

Pick and Assign

Once you've set up your NmrResidues in your HSQC (or other base spectrum), you will need to peak pick your 3D spectra. You could do this by going to Spectrum / Pick Peaks / Pick ND Peaks... (or type PP). Here you can select an area in which you peak pick your 3D spectrum. The problem with this approach is that you can end up peak picking lots of noise peaks or artefacts and you won't be able to transfer your NmrAtom assignments from your HSQC to the 3D. A better way is to use the Pick and Assign module.

Open this module by going to Assign / Pick and Assign or typing PA. Go to the module settings by clicking on the gear icon. Select the spectrum display in which you would like to pick your 3D peaks (make sure that you have a spectrum display module with your 3D spectra open already - it can also be helpful to have another display open with your HSQC/base spectrum). You can make changes to the peak picking tolerances here if you decide this is necessary at a later stage. In the main Pick and Assign module, select your NmrChain from the drop-down menu. You will see that the table is now populated with all of your NmrResidues in this NmrChain. It is also useful to open the NmrAtom Assigner which you can do by going to Assign / NmrAtom Assigner or typing AN.

Begin the process of picking and assigning your 3D spectra by double-clicking on an NmrResidue in the Pick and Assign module table. Your spectra will automatically navigate to the position of this NmrResidue, so in your 3D spectrum display you should see a strip of peaks ready to be picked. Peak pick these by clicking on Restricted Pick in the Pick and Assign module. Remember that peaks are picked at the currently visible contour levels, so make sure that your levels are such that you won't pick lots of noise peaks. It can also be helpful to switch off the negative contour levels for spectra where you are not expecting any negative peaks (go to right-click / Contours ... to do this). Now you can assign the H and N NmrAtoms from the HSQC to your newly picked peaks by clicking on Assign Selected. You can also do the peak picking and assignment in one go by clicking Restricted Pick and Assign.

Assigning the additional dimension

In the previous step, two the three dimensions of your 3D spectrum peaks were assigned to NmrAtoms. You can now also assign NmrAtoms to the additional third dimension. For backbone assignment spectra this will be typically be a carbon dimension, but it could also be a proton dimension if you are peak picking a 15N-NOESY-HSQC, 15N-TOCSY-HSQC, HBHAcoNH or other type of HHN spectrum.

To assign the carbon NmrAtoms for your backbone assignment spectra, select a peak (or several peaks which are all associated with the same carbon atom) and create a carbon NmrAtom by clicking on the CA, CB, CA [i-1], CB [i-1] or other assignment option in the NmrAtom Assigner. If you make a mistake, simply click on the same atom again to de-assign the peak(s) or directly click on the correct atom to replace the wrong assignment with the correct one. You can see that your carbon assignments have been made in the Pick and Assign module table and that new NmrResidues and NmrAtoms have been created in the sidebar. When you have assigned the carbon dimension of all your peaks you are ready to move on to the next NmrResidue. Double-click on a new NmrResidue in your Pick and Assign table and repeat the pick and assign process.

Remember that some peaks in your HSQC (e.g. tryptophan side-chain peaks) may not have associated peaks in the 3D spectra.

Automatic Assignment of Carbon NmrAtoms with a Macro

We have created a macro which will automatically assign the carbon NmrAtoms in HNCA/HNcoHA, HNCACB/HNcoCACB and HNCO/HNcaCO style spectra. First of all, make sure you set the Experiment Type for your 3D spectra. Then after the Pick and Assign step (with the newly picked peaks still selected) go to Macro / Run CCPN Macros / BBAssignCarbonNmrAtoms. Now check that the carbon NmrAtoms have been assigned correctly. For straight forward strips the macro should work well (including for Gly/Ser/Thr residues), but if there are overlapped peaks or unusual chemical shifts, then you may need to correct the assignments with the NmrAtom Assigner (shortcut AN). To make life even easier you can also link the macro to shortcut. The quickest way to do this is to go to Macro / Open CCPN Macros and the select the file. Then click on the Add macro to a shortcut icon (fourth one along in the toolbar) and either click Save and Close or move the path to a more convenient key combination first (such as q1, q2, w2 or w3).