Calculate 3J H-Ha coupling constants

Introduction

In Analysis, it is possible to calculate coupling constants from HNHA experiments. The principle behind this is to use the relative intensities of the amide diagonal peak and the amide-Ha cross peak to derive an estimation of the H-HA coupling constants. The coupling constants in turn can be used, to estimate the PHI protein backbone dihedral angle using a Karplus relationship. All calculations are done in the Data Analysis: 3J H-Ha popup, which is accessible through M: Data Analysis: 3J H-Ha Coupling.

Requirements

• • The experiment must be set to the Full Type “H{[N]+[HA]}”, which may be set in the {Experiment Types} tab of the Experiment: Experiments popup.

  • Peaks must be picked in the HNHA spectrum, for both the amide-Ha cross peaks and the amide diagonal peaks.

  • Peaks must be assigned to spin systems and to resonances in their amide dimensions.

Calculations

To calculate coupling constants, do as follows:

• • Go to the {Options} tab in the Calculate 3J[Hn,Ha] Couplings popup

  • Set “Peak List:”.

  • Set “Intensity Type:”. Be cautious in regions of the spectrum where peak overlap is significant enough to affect the size and shape of peaks. Where there is overlap using the peak “height” may perform better than “volume”, but this will not overcome the problem entirely. For these overlapping peaks, one may use the Peak Separator to get more accurate peak intensities and volumes.

  • Set the “Transfer Time:” (known from the pulse sequence).

  • Set the “Karplus Coefficients”. Changing the Karplus coefficients will affect the Karplus curve, which in turn affects the predictions of the PHI dihedral angles. The default values are commonly used for protein structures.

  • Set the “Only likely angles:” option. This option reduces ambiguity in PHI angle prediction by allowing only values that are in common regions of the Ramachandran plot.

  • Set the “Show Glycines:” option. No special provision is made for glycine residues, but they may be included in the analysis by choosing true for this option.

  • Set other parameters.

Now go to the {Spin System Table}, to see the results. 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 under |Intensity Ratio|, the estimated 3J HNHA coupling under |3J[H,Ha]| and the PHI backbone angles under |Φ Angles| (as estimated from the Karplus curve):

Preserving the data

There are three choices of how to preserve the data for posterity. The first is to make an NMR measurement list by clicking [Make Coupling List]. The second is to use the Karplus relationship to make a PHI angle dihedral restraint list by clicking [Make Dihedral Restraints]. The last option [Make Coupling Restraints] is useful if you have a protein structure calculation method that can back-calculate couplings and fit them to the experimentally derived values.