================================
Data Analysis : 3J H-Hα Coupling
================================


.. |pulldown| image:: ../images/pulldown.png
   :align: bottom


.. |check| image:: ../images/check.png
   :align: bottom


.. |radio| image:: ../images/radio.png
   :align: bottom


.. |float| image:: ../images/float.png
   :align: bottom


.. |int| image:: ../images/int.png
   :align: bottom


.. |entry| image:: ../images/entry.png
   :align: bottom


.. |button| image:: ../images/button.png
   :align: bottom


.. |ramp| image:: ../images/ramp.png
   :align: bottom


.. |selector| image:: ../images/selector.png
   :align: bottom


**Calculate 3J HN-HA Couplings Using an HNHA Experiment**

This system is designed to efficiently extract 3J coupling information from
the relative intensities of HA and amide peaks recorded in a quantitative HNHA
experiment. The couplings may be used, according to the Karplus relationship,
to estimate the Phi protein backbone dihedral angle.

The layout is broken down into two tabs: one for the results in the "Spin
System Table" and an other for setting the various options. Although the user
can adjust the options at any time the system will automatically perform the
calculations and fill the results table using the first HNHA peak list it
comes across.

For the calculation to be performed the experiment must be set to the specific
type "H{[N]+[HA]}", which may be set in the Experiment Types tab of the main
Experiments_ table. It is also assumed that a peak list has been picked in the
HNHA spectrum, for both the HA cross peaks and the homonuclear diagonal amide
peaks. For a peak within the selected list to be considered it must be
assigned to resonances in its amide dimensions *and a spin system*, for
example using the `Pick & Assign From Roots`_ tool. This spin system
assignment is the means to achieve the pairing of a diagonal amide peak with a
corresponding HA peak; if two peaks for the same residue are not in the same
spin system then the analysis will not be made. The peaks do not have to be
assigned to particular residues, i.e. they can have anonymous spin system
numbers. It is also unnecessary to assign the non-amide dimension (e.g. HA
resonance) of the peaks; the amide and HA peak are automatically distinguished
by sign and ppm location.

In the "options" tab the user can switch between different HNHA peak lists,
control how output data will be made; like where dihedral restraints of
coupling measurements go (which restraint set), and setup the parameters for
the estimation of Phi dihedral angles. Changing the Karplus coefficients, to
change the below curve, will affect the prediction of the Phi dihedral angle.
The user may wish to adjust this but the defaults are values commonly used
for protein structures.

In the main results table clicking on a particular row, assuming the follow
option and spectrum is selected, will cause the display to show the location
of peaks for the selected spin system. The 3J coupling values are
automatically extracted from the relative intensities if the HN (diagonal) and
HA peaks, according to the method referred to below, whenever the table is
updated. The coupling measurements may be saved in the CCPN project by using
[Make Coupling List] - this creates a kind of measurement list which may be
inspected at any time, without having to repeat any calculations. Making
"Coupling Restraints" is specifically for 3D structure calculations that know
how to interpret such information. The dihedral angle estimates that are
presented are made according to the Karplus curve displayed in the "Options",
noting that because of its oscillatory nature there may be more than one range
of possible angles for a particular 3J coupling. The "Only likely angles"
option can reduce the ambiguity in Phi angle prediction by allowing only
values that are in the common regions of the Ramachandran plot. Angle
predictions may be converted into dihedral angle restrains, for use in
structure calculations, with the [Make Dihedral Restraints] button.

**Caveats & Tips**

Because this method relies upon peak intensities, the user should 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.

Residues may be excluded from restraint and coupling list generation by
double-clicking in the "Use?" column.

If a residue or spin system appears to be missing, check that the correct peak
list is selected (there could be many for one spectrum) and that both peaks
for a residue are assigned to a spin system in their amide dimensions.

No special provision is made for glycine residues, but they may be included
in the analysis using the "Show Glycines" option.

**Reference**

The method used by this system closely follows that which is described in the
following reference:

*G. W. Vuister and A. Bax (1993). "Quantitative J correlation: a new approach
for measuring homonuclear three-bond J(HNHa) coupling constants in
15N-enriched proteins". J. Am. Chem. Soc. 115 (17): 7772-7777*

.. _Experiments: EditExperimentPopup.html
.. _`Pick & Assign From Roots`: LinkPeakListsPopup.html



Main Panel
==========

|button| **Clone**: Clone popup window

|button| **Help**: Show popup help document

|button| **Close**: Close popup

Spin System Table
=================

*Documentation missing*

==================  ==================================================================================================
**Table 1**
----------------------------------------------------------------------------------------------------------------------
*Root Spin System*  Assigned name of the root (amide) spin system 
            *Use?*  Whether or not to use a given spin system when recording restraints & couplings  *(Editable)*
        *3J[H,Hα]*  The three bond amide H to alpha H coupling in Hz 
  *Error 3J[H,Hα]*  The estimated error in the three-bond HN-HA coupling 
            *ΔδHα*  Difference between the observed and (sequence adjusted) random coil HA chemical shift 
        *Φ Angles*  Predicted phi backbone torsion angles, in degrees, according to the Karplus equation 
 *Intensity Ratio*  Ratio of peak intensities used in calculation: HA crosspeak over diagonal 
 *Amide Intensity*  Intensity of amide H diagonal peak 
     *α Intensity*  Intensity of alpha H crosspeak 
==================  ==================================================================================================



|button| **Assign non-root dimensions**: Assign amide H and alpha H resonances to the indirect 1H peak dimensions

|button| **Make Coupling List**: Make a J-coupling measurement list from results; stored in CCPN project

|button| **Make Dihedral Restraints**: Make a dihedral angle restraint list from results

|button| **Make Coupling Restraints**: Make a J-coupling restraint list from results

|button| **Update Table**: Manually update the table & redo calculations; useful after adjusting intensities etc.

Options
=======

*Documentation missing*

|pulldown| **Peak List**: Selects the HNHA peak list to calculate couplings from

|pulldown| **Intensity Type**: Selects whether to compare peak heights or volume integrals

|float| **0.01305**: The transfer period used in the HNHA experiment

|float| **1.11**: Correction factor to compensate for differential relaxation

|pulldown| **JCoupling List**: Selects a J-coupling list to store results in

|pulldown| **Restraint Set**: Selects a restraint set to make dihedral (phi) angle restraints in

|pulldown| **Window**: Selects a spectrum window to display peak locations in

|check| **Show Glycines**: Whether to show Glycine residues in spin system table: defaults off due to two HAs

|check| **Mark Peaks**: Whether to mark peak positions in windows with lines

|check| **Follow Peaks**: Whethe rto follow peak positions in a spectrum window when clicking in the spin system table

Φ Dihedral Angle Prediction
~~~~~~~~~~~~~~~~~~~~~~~~~~~


|float| **6.51**: The first coefficient in the Karplus equation; for the cosine^2(angle) term

|float| **-1.76**: The second coefficient in the Karplus equation; for the cosine(angle) term

|float| **1.6**: The third coefficient in the Karplus equation; the scalar constant

|check| **Only likely angles**: Whether to show only predicted phi angles for common protein conformations

|float| **30.0**: Width to derive upper and lower bound for dihedral angle restraints

Karplus Curve
~~~~~~~~~~~~~




