Assigning side chain resonances

In the previous section it is explained how spin systems can be scored and assigned to spin system or residue types. However, in order to uniquely assign a spin system to a residue type, or to increase the reliability of the spin system typing, it is necessary in most cases to obtain all the side chain resonances of a spin system.

Even if not absolutely necessary at this stage, having a full set of side chain resonances assigned to the atom types and spin systems assigned to spin system types will simplify linking of sequential residues, e.g. in case of ambiguities due to overlap, and mapping of stretches of sequentially linked spin systems on the primary sequence. Furthermore, side chain assignments are essential in verifying sequential assignments and needed for a detailed structure analysis.

The scheme we follow here is:

• • identify the intra residual HA, CA and CB resonances

  • if a sequential connection is known, identify the HB resonances from the HBHAcbcacoNH

  • navigate in the hCCH-TOCSY to the HA - CA position

  • analyse the hCCH-TOCSY pattern found at the HA - CA position to obtain carbon frequencies of side chain carbons

  • navigate to these carbon frequencies in the hCCH-TOCSY and search for the identical patterns

  • when an identical pattern is found, make an assessment of the spin system type and assign 13C and 1H  resonances to atom types.

We start with spin system {42}. Using the spin system typing based on the CA and CB shifts described in the previous section, spin system {42} is most likely an Ile, with much lower probabilities for Tyr, Phe:

To obtain the HA frequency from the HNcaHA spectrum, navigate to spin system {42} in the HHN window using:

• M: Resonance: Spin Systems

  • In Resonance: Spin Systems:

    • select spin system {42}

    • click [Display Strips] with window HHN selected in upper right pulldown menu

  • Use the HBHAcbcacoNH experiment to verify that the HA of {42} is at 3.535ppm:

Select the peak at 1H: 3.535 ppm and mark it. Open Assignment: Assignment Panel (R:Assign:Assign HNcaHA:HNcaHA....), set the resonance to atom type HA and add it to spin system {42}.

Note: checking the residue type score now will give a slightly different result, due to the extra information that is now present for this spin system.

Now that we have the CA and HA combination of {42}, we navigate to the corresponding position in the hCCH-TOCSY to check which 13C side chain resonances {42} has. For ease of use, we set the maximum number of marks to 4, using Window: Marks and Rulers (M: Window: Marks and Rulers). In the HCN window, mark the peaks corresponding to the CA and CB resonances of {42}:

Select the CA peak, and use it to navigate to the hCCH-TOCSY: R: Navigate: 1H - 13C in HCC. In the HCC window you will find now the pattern of side chain resonances belonging to spin system {42}, with the HA, CA and CB frequencies marked (in case the window didn't focus on the marked resonances, use the scroll bars on the bottom of the window to center manually):

The hCCH-TOCSY pattern at the HA-CA frequencies (x-axis 3.535ppm, z1-axis 65.74ppm) shows the CA and side chain carbons along the vertical y-axis. In this case the pattern that we observe is typically a Ile pattern, it is not overlapping and we could assign this spin system confidently to a Ile:

All we need to do now is to verify the pattern of resonances in the hCCH-TOCSY at the carbon frequencies that we found at the HA-CA position of {42}. To do this, we use the option to strip spectra in Analysis. We count 5 peaks, corresponding to CA, CB, CG1, CG2 and CD1. To pick these peaks, find the maximum of the intensity first by moving the view 1 plane to 65.58ppm and pick the peaks using 'CTRL'+'SHIFT' and draw a box around the peaks (Note: if the peak picker fails to pick all of them automatically due to overlap in the z-dimension, add peaks manually where needed). After picking the peaks, assign all the peaks to atoms HA - CA of spin system {42}.

To search identical patterns at the CB, CG1, CG2 and CD1 frequencies we add 4 strips to the current view in the HCC window, which are all displayed at the same CA frequency in the z-direction:

• • click [Strips]

  • click [+] 4 times:

In order to navigate within in a single strip to a different z-location do the following:

• • activate the strip that you want to navigate in. For example, by clicking [2], you activate strip 2.

  • navigate to the desired z-position by using R: Navigate: 1H (F3)13C (F2)13C in HCC at a chosen cursor position in the spectrum, or by selecting a peak at that position.

After the strip has navigated to the correct position, panning in the x-direction is usually needed to find the pattern we are interested in (which is at the 1H frequency of the attached proton). Here is an example to clarify the procedure:

To find and display the hCCH-TOCSY pattern originating from the HB-CB do the following:

• • activate strip 2

  • in strip 1, select the peak corresponding to the CB at 38.489 ppm

• Use R: Navigate: 1H (F3)13C (F2)13C in HCC

  • in strip 2, move the focus of the spectrum until you find a pattern identical to the one in strip 1. You will find this at 1.59ppm, the HB frequency.

  • Find the maximum intensity of the train of peaks in the z-direction and pick the peaks.

  • Select the peak that is located at the HB-CB-CB position and assign it to HB - CB - CB of {42}. Note: you need to create a new resonance for the HB frequency, and add it to the same spin system. 

Your display should now look something like:

Now that you have found the HB, you can propagate the assignment of the HB - CB to the rest of the hCCH-TOCSY peaks at the HB-CB position, although this is not necessary.

Repeating this procedure for the rest of the observed carbon frequencies, we can identify all the carbon and attached proton frequencies of this Ile residue. Where needed, create new resonances and assign them to the same spin system.

Note that the CG1 has 2 attached protons, and thus at the CG1 frequency in the z-direction we find 2 identical patterns, each belonging to 1 of the 2 HG1 protons! Look in the residue information, then it's clear what to expect (M: Molecule: Residue information).

Using M: Resonance: Spin System Types, you can now assign spin system {42} to Ile.

The procedure described above can now be repeated for all the spin systems found in the spectra in the HCN and HHN windows. Please note that some side chains cannot be assigned unambiguously at this stage due to overlap. Just like side chain assignments can help in resolving ambiguities in sequential assignment, sequential connectivities can help in resolving ambiguities in side chain assignments. The sequential connectivity can identify the residue type based on information from neighboring residues and primary sequence, and thus can help searching for the correct patterns in experiments that reveal side chain resonances, such as the hCCH-TOCSY used here.

The project with {42} assigned is saved as NapD_6. The project includes unassigned hCCH-TOCSY peaks.

Chris Spronk: Note that these peaks have been imported from the easy project that was used for the publication of the NapD structure. The peak positions of the unassigned peaks are sometimes slightly off. This will be changed later.

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