Scalar couplings

Introduction

In the next part of this tutorial we shall look at a different kind of data extraction. In this case we will look at extracting coupling constants from the quantitative HNHA experiment. The basic principle here is that by measuring the relative intensities of the amide diagonal peak and the amide-Ha cross peak this special experiment gives us an indication of what the H-HA coupling constant (and hence the backbone PHI angle of residue) might be.

Calculating scalar couplings

Firstly have a look at window4. You will see that we have already picked many of the HNHA peaks, including the diagonals, and have assigned them to the amide resonances. This was achieved using the M: Assignment: Pick & Assign From Roots functionality, as described in the previous tutorial. Note that we did not have to explicitly assign indirect proton H and HA resonances (Y axis for this window).

Bearing this setup spectrum in mind, go to M: Data Analysis: 3J H-Ha Coupling. The first notable thing when the popup appears is that the main table is already filled in. This is because the experiment type is already set correctly to "H{[N]+[HA]}". You can go to M: Experiment: Experiments, {Experiment Types} to verify this:

In the Calculate 3J[Hn,Ha] Couplings popup select the {Options} table and have a look at the settings that have been used to fill in the main table. Specifically note that you are required to have specified the right "Transfer Time:" for the experiment (known from the pulse sequence) and have reasonable estimates for the Karplus Coefficients. Calculating such coefficients with great precision is not something we will go into here.

Returning to the {Spin System Table} note that by selecting on a residue's row you are instantly zoomed to the corresponding amide location in the selected window, so that you can investigate any peaks. If you wish to exclude a particular residue from being used to make any output you can simply toggle (double-click) the |Use?| column to "No". The analysis of the amide-HA/amide-amide intensity ratio can be seen and the estimated 3J HNHA coupling and PHI backbone angles (as estimated from the Karplus curve) that derive from this are filled in. Of course the accuracy of such estimates very much depends on the parameters you have entered and whether there are any peak distortions or overlaps which would give unrepresentative intensities.

When you are confident with your values, peaks and peak selection you have three choices of how to preserve the data for posterity. The first is to [Make Coupling List] - this simply makes an NMR measurement list (in the same vein as chemical shifts or T1 rates). The second is to use the Karplus relationship to make a PHI angle dihedral restraint list for your protein which can be used in structure calculations: click [Make Dihedral Restraints]. The last option is to [Make Coupling Constraints] which is useful if you have a protein structure calculation method that can back-calculate couplings and fit them to the experimentally derived values.

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Next: Dihedral constraints