Data Analysis : 3J H-Hα Coupling

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

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

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