Theoretical Aspects

Summary

How to use MODULE
 

Required Material

Starting the Program

Input File Format

Initial Display

Displaying the Primary Sequence

Selection of Type of Coupling

Setting Internuclear Distances

Fitting Single Modules

Fitting the Alignment Tensor

Correlation Plots

Comparision of Calculated and Experimental Couplings

Monte Carlo Error Analysis

Test Sample 1

Choice of Modules from Primary Sequence

Fitting the Alignment Tensors

Correlation Plots

Comparision of Calculated and Experimental Couplings

Monte Carlo Error Analysis

Common Alignment Frame

Degenerate Orientations

Automatic Calculation of Molecular Architecture

Covalent Bonds

Test Sample 2

Special Cases

Axially Symmetric Alignment Tensor.

Highly Rhombic Alignment Tensor.

 
Distance Constraints

Creating and Reading Work Folders

Simulating Datasets

Hammerhead Ribozyme - Orientation of Secondary Structural Motifs.

Test Sample 3

Protein-Protein Complexes.

Test Sample 4
 
 

One of the main uses of MODULE, in our hands at least, is the ability to fit multiple different modules and treat them as rigid oriented objects in a common reference frame of the alignment tensor, this part of the manual will explain how to do this and illustrate some of the features we have included to help treat this kind of orientational molecular modelling.
 

Choice of Modules from Primary Sequence

The regions of primary sequence to be used for the different modules can be selected using the cursor once the pdb file has been read : In this case two helices and the central beta sheet of our model system have been selected as representing different regions of known structure. The rest of the molecule represents the third module (yellow).
 
 

Note that the 2nd Display can be used to visualise the molecule while selecting the regions from the primary sequence.

The Select button on the bottom left of the interface allows residues or atoms to be identified with the cursor.
 
 
 

Fitting the Alignment Tensors

To fit the tensors to the selected couplings click on Fit in the main MODULE window - This should take a few seconds per module on a R10000 SGI processor (approximately equivalent to a 400MHz PC)
 
 

Having fitted the tensor, the axial and radial components (A_a and A_r) and the euler angles describing the orientation of the tensor relative to the pdb frame, are shown in the background window.

The tensors can then be visualised with respect to the pdb coordinates (Orientation in the Visualisation menu) -
 
 

Correlation Plots

To inspect the quality of the fits, or to identify outliers, correlations of calculated and experimental couplings can be viewed, either separately or for all couplings together by selecting the option Back-Corr in the Visualisation menu:
 
 

Selecting Separate Couplings gives the correlations between calculated and experimental for each different type of coupling, defined by the different atom types.
The data from each different module are coloured with respect to the colour of the particular module M1-M6.
 
 

The correlation for each particular coupling type can be viewed in detail using the same menu. The cursor can be used to identify the specific couplings.

Note that in each of these options the local chi2 is shown with respect to the individual points on the x-axis. This allows outliers or problematic couplings to be identified easily. A text line in the window gives the name, chi2 and calculated and experimental values of the coupling corresponding to the cursor poisition (yellow line).

In the example shown below we have selected the CA-CO :
 
 

Similarly, the experimental and calculated couplings can be visualised with respect to the primary sequence, either separately or for all couplings together by selecting the option Back-Diff in the Visualisation menu:
 
 

Again the data from each different module are coloured with respect to the colour of the particular module M1-M6.
 

Separate Couplings -

HN-N Couplings

Monte Carlo Error Analysis

The uncertainty associated with the orientation of the tensor axes, and the values of the axial and rhombic components can be estimated using a Monte-Carlo based error analysis.

This analysis takes the best-fit tensor and back-calculates a simulated datset for each set of couplings. A gaussian noise distribution is then centred on these values, based on the uncertainty in the initial input data file.

The Monte Carlo analysis can be applied to all, or only one single module, and is selected using the button in the main display window.
 
 

As described above the dispersion in the magnitude of Da and Dr can be visualised using Monte Carlo in the Visualisation menu