stc Brian Sykes Lab

Table of Contents

1. Introduction
   • Latest news from the stc website
   • Overview
   • References, Acknowledgements and Copyright
   • Download and Installation

2. Using the Software
   • Setup your Data
   • The Initial STC Window
   • The Calc ASA Window
   • The Thermodynamics Window
     • Output - Basic results
     • Output - Verbose results
     • Output - Histograms
   • Multiple Structures
   • Multiple Structure Scripts
   • Troubleshooting

1. This software is a self contained tcl/tk starkit which should make installation fairly trivial. See the Release notes for the latest updates. Download the software appropriate for your machine:
   • MacOSX - Mac (intel)
   • MacOSX - Mac (powerpc)
   • Linux x86 - Intel x86 32 bit
   • Linux x86_64 - Intel x86 64 bit

2. Macosx - Double click the download file. You should now get a new file on the desktop (or your download directory) represented by the stc icon. You should be able to double click the icon to run the program.

   Linux - Same as the macosx procedure but you may have to drag the directory from the opened window to the desktop. If this is not working then use a terminal command like:

   	tar xvf stc*.tar
   

   to get the stc directory and then move it to the desktop.

3. Next, let's make sure the software is working and we understand the data files required. Download data and examples (11MB) and follow the procedure above to create the stc-6.0-x-examples folder. (see the examples.readme file for a quick summary of each example provided).

4. To run an example, double click on the stc.command file packaged within each example directory. For linux, you may get a dialog asking what to do next, just click on the "Run" button.

5. Finally, if you wish to keep the software in a more permanent location, pick one of the methods below:
   • For macosx, create /Applications/bdslab and drag the stc icon and the stc-examples here.
   • For macosx, drag the stc icon into /Applications.
   • To install in /usr/local or some other third party software repository, execute terminal commands similar to the following:

     # Move (or remove) any previous installations of stc. mv /usr/local/stc /tmp/stc.old; rm /usr/local/bin/stc # Move the software to an appropriate place in /usr/local. mv $HOME/desktop/stc /usr/local/stc # Make a link in /usr/local/bin to the executable. ln -s /usr/local/stc/stc /usr/local/bin/stc

1. Create a working directory.
2. Copy structure files here (pdb format).

3. (optional) Create an stc.defaults file to customize how this software runs.
4. (optional) Copy a custom residue library here.
5. (optional) Copy each custom ASA file here.
6. (optional) Copy each custom sconf file here.

Note that the system type for our data is "binding". One could also choose "unfolding" or "oligomerization". See the examples.readme file for examples involving the other system types.

The output directory is where stc writes all the output files. If you start stc by clicking on the icon, the default output directory is $HOME/Desktop/stc_out. If you start stc via the command line, then the default output directory is ./stc_out. You can automatically set the output directory in the stc.defaults file or within the gui.

The residue library is where we define the amino acids, nucleotides, and parameters used in the calculations. Users with unique amino acids will need to create a customized residue library. You can automatically set the residue library in the stc.defaults file (see amino_def resource) or within the gui. See the xuw example we have provided.

The Calc ASA button allows one to calculate accessible surface area (ASA) output files. The Thermodynamics button lets the user make thermodynamic calculations based on the ASA files. Batch is a special case where the user wants ASA and thermodynamic calculations to be applied to several pdb data files.

Most users start by clicking on the Calc ASA to create the accessible surface area files. When that has been setup, press on the Thermodynamics button.

A results window is displayed which allows you to view the various output files. All accessible surface area output files have the ".acc" file suffix. The log file is important because it indicates all the steps and programs used to derive the ".acc" files and it indicates the nature of any errors. Here are examples of an acc file and a log file.

When you are done, click on the "Dismiss" buttons until you arrive back at the first stc window.

In this window we make thermodynamics calculations based on accessible surface area files. Note that stc fills in the "ASA file" fields but the user can create or edit his/her own. The other fields are set such that stc calculates sConf in a reasonable way.

Although stc will calculate "sConf" automatically, the user has several options in determining how it is calculated. First note that calculating sConf for each residue is the summation of the individual entropy components "ΔSbu->ex", "ΔSex->u", and "ΔSbb". ΔSbu->ex is calculated from the amino acid table . ΔSex->u and ΔSbb are normally zero unless the user has specified residue ID numbers for residues that remain unfolded. In this case, these components are taken from the amino acid table.

Some users have experimental values for ΔSbb or ΔSex->u. In this case an optional sconf file allows the user to specify values of ΔSbb and ΔSex->u for each residue. You must click the "calculate from datafile" button in order to use these values or "estimate" which weights the ΔSex->u values according to total sidechain ASA of the molecule.

Click the Calculate button and see the next three sections for a discussion of the results.

The program displays a window which shows the basic output file of various binding constants and key variables.

Section 1-ASA (Accessible Surface Area) values

This section defines the polar and nonpolar ASA values for the free ligand, bound ligand, free enzyme, and bound enzyme. These values are presented in a table 1. The "free ligand ASA - bound ligand ASA" and "free enzyme ASA - bound enzyme ASA" for both polar and nonpolar quantities are what STC uses to calculate the thermodynamics of binding. All changes in ASA are given in Section 2, Table 2.

Table 1
Species     Polar ASA (A2)      Nonpolar ASA (A2)
Free Ligand         #               #
Bound Ligand        #               #
Free Enzyme         #               #
Bound Ligand        #               #

Section 2-Change in ASA values
This section defines the change in ASA, nonpolar and polar, when comparing the free verses bound ligand and free verses bound enzyme. The difference in polar area upon binding for the ligand is then added to the respective polar value for the enzyme. This total change in ASA polar is what STC directly uses for thermodynamic calculations. The difference in nonpolar area upon binding for the ligand is then added to the respective nonpolar value for the enzyme. This total change is ASA nonpolar is what STC directly uses for thermodynamic calculations. These are global values, the ΔASA for individual atoms and residues are listed in the detailed output file Tables 1-4.

Table 2
 Species     Polar ΔASA (A2)     Nonpolar ΔASA (A2)
 ligand               222.30               740.50
 enzyme               513.14               770.96
 ligand+enzyme        735.44              1511.46

Section 3-Thermodynamic values
This section defines the thermodynamic values calculated from the ΔASA nonpolar and ΔASA polar contributions from the ligand + enzyme. The thermodynamic calculations listed are the global values, the individual atom or residue values are listed in the detailed output file. For more details please look up our references and acknowledgements.

ΔCpbind is the heat capacity of binding and should be a negative value (positive if viewed as dissociation). This value is calculated from the ΔASA nonpolar and ΔASA polar values. The unit for heat capacity is kcal/mol/K.

ΔHbind is the enthalpy of binding and can be positive or negative. If it is positive then the enthalpy of binding is unfavorable, if negative then the enthalpy of binding is favorable (the opposite is true for dissociation). The enthalpy of binding is calculated from the heat capacity of binding plus the binding enthalpy at a reference temperature of 60 degrees C. The unit for enthalpy of binding is kcal/mol.

ΔSbind is the entropy of binding and can be positive or negative. If this number is positive then the entropy of binding turns out to be favorable, if negative then the entropy of binding turns out to be unfavorable (the opposite is true for dissociation). The unit for entropy of binding is kcal/mol/K. The entropy of binding is a sum of 3 entropies that are listed below. The first is the solvation entropy, ΔSsol, which is directly calculated from the ΔASA nonpolar and ΔASA polar values (unit is kcal/mol.K). The second is the overall rotational/translational entropy, ΔSrt, which has a set value of -0.008 kcal/mol.K. The third is the conformational entropy, ΔSconf, which in itself is a sum of 3 entropies. The first contribution to conformational entropy is ΔSbu_ex, which is the change in conformational entropy of the side chains due to tertiary or quaternary interactions during binding. The second contribution to conformational entropy is ΔSex_u, which is the change in conformational entropy of the side chains due to secondary structure changes upon binding. The third contribution to conformational entropy is ΔSbb, which is the change in conformational entropy of the backbone upon binding. The 3 types of conformational entropy are generally unfavorable with respect to binding resulting in a negative value having the unit kcal/mol.K. The overall conformational entropy of each residue is given in Table 4 in the verbose output file. The individual contributions (ΔSbu_ex , ΔSex_u, and ΔSbb) to conformational entropy per residue is also given in Table 4 in the verbose output file.

TΔSbind is the entropy of binding at the temperature where the structure was determined. This value is useful in calculating free energy.

ΔGbind is the free energy of binding and it is calculated from ΔHbind - TΔSbind. A negative value for ΔGbind indicates binding is favorable, a positive value for ΔGbind indicates binding in not favorable. From ΔGbind one can quickly calculate a Ka and Kd. Many of these thermodynamic values are given for each residue in Table 5 in the detailed output file.

If you click on the verbose output file you can see thermodynamic output on specific atoms or residues. Here is where you can study the individual atom differences, the classification of polar/nonpolar atoms, a residue by residue summary in the calculation of sconf (including the change in ASA for total polar and non-polar atoms) and the calculation of themodynamic paramaters per residue.

Section 2: Individual Atom Differences in ASA

This section displays the results for atoms having a change in their ASA when comparing the free ligand with the bound ligand and free enzyme with the bound enzyme. The first table shows the results for the ligand, the second table is for the enzyme. Only atoms with a change in ASA are shown.

 ResId  Name   Atom     ΔASA  Definition 
                         A2                 
     1   ALA      N   0.2363  polar
     1   ALA     CA  11.3143  nonpolar
     1   ALA      C   0.1231  nonpolar
     1   ALA      O   0.1229  polar
     1   ALA     CB  12.8967  nonpolar
     2   LEU     CA  -0.3348  nonpolar
     2   LEU      C   0.5131  nonpolar
     ...

Section 3: Amino Acid Differences in ASA

This section displays the results for residues having a change in their ASA when comparing the free ligand with the bound ligand and free enzyme with the bound enzyme. The first table shows the results for the ligand, the second is for the enzyme. Only residues with a change in ASA are shown.

ResId  Name        ΔASA Polar      ΔASA Nonpolar
                        A2                A2
    64   ASP          0.8498             0.0000
    66   HIS          3.2483             0.0000
    92   ASP          1.3114             0.0000
    96   ARG         59.3139             8.5508
   124   ASN          3.2231             0.0000
   ...

Section 4: Calculating ΔSconf

This section displays the calculation for the conformation entropy for each residue. This is done separately for the ligand and the enzyme. It also includes the ΔSbu->ex, ΔSex->u, and ΔSbb values used to compute ΔSconf per residue. ΔASATot is the sum of nonpolar ΔASA and polar ΔASA for each residue. ΔASAsc is the change in ASA for the side chain of each residue. The next 3 entropy values deal with structural changes in side chain or backbone conformations upon binding. When added together they total ΔSconf.

ResId  Name   ΔASATot    ΔASAsc  ΔSbu->ex    ΔSex->u    ΔSbb       ΔSconf  
                 A2         A2   cal/mol.K   cal/mol.K   cal/mol.K   cal/mol.K
     1   ALA   24.6933   12.8967    0.0000     0.0000      0.0000      0.0000
     2   LEU   68.4463   68.0124    0.8092     0.0000      0.0000     -0.8092
     3   MAS   26.6067   26.7675    0.0000     0.0000      0.0000      0.0000
     ...

Section 5-Thermodynamic values for each residue

This section gives the heat capacity of binding, the enthalpy of binding, the different entropies of binding, and the free energy of binding for each residue. The entropy values are multiplied by the temperature the structure was calculated at (oK).

ResId  Name    ΔCpbind     ΔHbind    TΔSsol   TΔSconf    TΔSbind     ΔGbind 
               kcal/mol.K  kcal/mol  kcal/mol  kcal/mol  kcal/mol  kcal/mol
     1   ALA    -0.0109     0.5741    0.8327    0.0000     0.7581   -0.1840
     2   LEU    -0.0306     1.6392    2.3402   -0.2413     2.0244   -0.3852
     3   MAS     0.0041    -0.8211   -0.0691    0.0000    -0.1437   -0.6774
     4   ARG     0.0046    -1.8806    0.3116   -0.6848    -0.4477   -1.4328
     ...

You have several structure files and you want to see which one has the most favorable thermodynamics. This window allows the user to do this quickly.



First you want to place all of your pdb files in one directory as shown in our example data directory stc-examples/batch.gui. In this example, the pdb files are made up of a ligand and an enzyme separated by a TER statement. When you are ready, press the Batch button on the main stc window. Currently there are not alot of options, the most interesting being that you can choose which variables you want to display as the results are being calculated. Your choice of variables come from the typical basic output file .

After pressing Calculate, each pdb file is processed in alphabetic order. ASA output files are generated and thermodynamic calculations done with default parameter settings. A one-line summary of the thermodynamic results are shown on the screen and sorted results are also available in the output file stc.batch results . To see the thermodynamics details for any particular pdf file, just click on the line containing the summary results.

If you wish to bypass the gui altogether and are comfortable with writing scripts, then take a look at the batch_stc file in the examples directory. Several users have expressed an interest in using routines within this package in ways beyond the scope of this software.

Here are some notes for stc script writing. Thermo is the independent back-end of stc and a user could type thermo and be prompted for all the input files required to perform the calculations. Thermo produces two output files, a short version which stc displays in a window and a verbose file.

Getting input files ready for thermo is a fairly complex chore in itself. A shell script called calc_asa takes the user's input pdb files and calculates the accessible surface area files needed by thermo. The main program to do this is called access which is a fortran program also used in vadar.

Getting pdb files into acc format involves the following.

1. fix_pdb, check and reformat pdb files. Discrepancies in naming convention are delt with here.
2. convert pdb format to diamond format. (pdbtodia)
3. use a program modified by William Wilcox to add standard van der waals (addradii).
4. modify the addradii output to have van der waals which match the amino acid library instead (edpdb).
5. call access with edpdb output to calculate accessible surface area.

Here is a common problem in the stc.log file:

1. *** Error, Unrecognized residue name:     577 S1  EMD     1     61.88000
   1.41900   0.21200      1.8000
   Define this residue in the amino.def file if vanderwaal assumptions are wrong
   

   One or more user pdb files contains an amino acid that STC does not recognize. See the stc-examples/xuw directory where there is an example of how to modify the amino.def file.

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