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ANALYSIS OF INTEGRAL 

CHARACTERISTICS

[Tutorial page]

 

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Main goal
      As the integral protein characteristics, the summarised charge Q+ of a group of residues in the region of interaction of alpha-helices H1 and  H2 of homeodomain (in terms of isoelectric point value) is considered.

The package CRASP estimates:

-  the impact of co-ordinated substitutions into the constancy of Q+ value
-  the relationships between charges of residues at different helices, that is, between QH1 (summarised charges at positions L13, R15, R18, E19,N23) and QH2 (summarised charges at positions R30, Q33, S36, E37).
-   constancy of the difference between QH1 and QH2 (Q-=QH1 - QH2)

Denotations:
Q+ - summarised charges at positions L13, R15, R18, E19,N23, R30, Q33, S36, E37
QH1 - summarised charges at positions  L13, R15, R18, E19,N23
QH2 - summarised charges at positions  R30, Q33, S36, E37
Q+=QH1+QH2
Q-=QH1-QH2.

Preliminary step
Open new browser window integral characteristics analysis CRASP page. This will be the CRASP package working window. Use it in parallel with this tutorial window. Open new browser window Result example page to retreive the data from test analysis.

The description of analysis, methods and algorithms could be obtained here.

Step 1. Input the sequence data
Enter the sequence alignment data in FASTA format into the 'Sequence data' field or download the sequence data from file by using dialog window 'Load from file'. Click the button 'ON' and input the file name into the text field.
This is an image of the CRASP sequence data input section
Your actions with the working window:
  • Load the page with the homeodomain sequence alignment in result example window. Mark all the sequence data and copy it in clipboard.
  • Return to your working window. Set 'Load from screen' button ON and paste the sequence data into the text-box below.

Step 2. Input calculation parameters
Select calculation parameters in drop-down menus and editable text fields:
input_par1i.gif (2519 bytes)
Select physico-chemical residue's characteristics from the 'AminoAcid quantity' menu, which contains 36  properties.
Important note: this option is valid if 'AAindex number'  field contains zero value.
input_par2i.gif (9601 bytes)
You may select one of more than 400 characteristics from AAIndex database (see details). Type the database entry number.
input_par3.gif (1216 bytes)
In CRASP, Monte Carlo technique is applied to estimate the statistical significance of the constancy of the chosen physico-chemical parameters. For this purpose, a large number of random samples with independent amino acid substitutions in alignment positions is generated and folowed by subsequent evaluation of statistical parameters of dispersions of integral characteristics in these samples.  The number of samples generated is ordered by menu 'Random samples number'.  For the characteristic including many positions and for huge sequence samples, the calculations are time-consuming, so we recommend to use small values for this parameter.
input_par3i.gif (3560 bytes)
Your actions with the working window:
Set the following calculation parameters:
  • Select in the field 'AminoAcid Quantity' the option 'Isoelectric point'
  • Select in the field 'AAindex number' the option '0'
  • Select in 'Random samples number' the value '500'

Step 2. Input weighting data
Sometimes it is necessary to apply data weighting. CRASP package realises several standard schemes of data weighting. Also, a user may enter other weights for all sequences. Data weighting is set in the fields below.
wt_input.png (17487 bytes)
Current version of CRASP applies several weighting methods. Select one of them or the option without weighting 'Off'. By choosing 'User defined' weights, enter weight values for each sequence that should be divided by symbol-separator (by default - ;). You may introduce your own symbol-separator, specified in the text-box 'Separator'. By using the weights by Altschul et al. and Felsenstein, enter the phylogenetic tree in *.ph format or load it from file. Details are here.
wt_options.gif (3738 bytes)
Your actions with the working window:
  • Select the weighting method:
    • Felsenstein
  • Go to the page with homeodomain phylogenetic tree in result example window. Mark the data and copy in clipboard.
  • Return to your working window. Set [Load from screen] button ON and paste the data into the text-box.

Step 3. Integral characteristics setup
It is possible to introduce up to 4 different integral protein characteristics that are related to the chosen physico-chemical property of residues. In the field 'Name' enter the name of characteristic. In the field 'Description', enter numerical coefficients for each protein position which influences the characteristics selected. Coefficients are ordered in a special format. See details here.
input_integrals.png (10061 bytes)
Example of integral characteristics setup.
input_par5i.gif (3972 bytes)

Step 4. Output parameters setup
Select the format of output data. The data could be displayed in a text form or as GIF-images. By clicking the check-boxes, it is possible to display information about distribution of characteristic values in original sample (first column), about dependence between two integral characteristics (second column), about distribution of characteristic variances in random samples (third column), about distribution of dispersion ratio in random samples (fourth column).
foutput.png (10147 bytes)
Your actions with the working window: 

Set up the following parameters:

fcparam..gif (8841 bytes)

Step 5. Running CRASP
To RUN CRASP click the button 'Execute':
runf.png (5598 bytes)
 

Your actions with the working window:

  • click 'Execute'
The  result page will be displayed automatically.