HyPare

Output Sample

The HyPare output consists of four main parts:

In the following example we will address the complex 1jtg (TEM complexed with its inhibitor BLIP). Our goal is to find hotspots for assoication for the complex at a salt concentration of 0.150 M. Hotspots are those residues that significantly alter the association rate. HyPare uses a cut off point of 4 fold. This is what our input would look like:

Note that the experimental association rate and salt concentration are the default ones. In finding hotspots only the target salt is important, even if you do not know the experimental data. After clicking on calculate, the program starts to work. When done, this is what we get as our output:

So how do we really understand what we see? Lets take it one by one:

Summary Table  

As you might guess, this table summarizes the input parameters that you have enetered, as well as some important numbers about your complex.

  1. Each file processed appears in a different row. In our case, there is just one, 1jtg.pdb.
  2. Next we see the coulombic energy, -1.62 kcal/mol, as it was calculated for 0.022 M salt.
  3. Note how the energy moves up at the higher salt concentration of 0.150 M. consequently, the rate of association drops to 7.15e+04M-1s-1 from the experimental value of 1e5 M-1s-1.
  4. The basal rate is found to be 1.65e+04M-1s-1. This is the association rate with no electrostatic contribution.
  5. The last two columns shows the total charge found for each chain and for the complex.
  6. clicking on export files will take you down to the download section (see below).
Residue Analysis  

This section provides a residue by residue analysis of your complex. HyPare performs two mutations at each residue location. In the first, it assigns a positive charge, and in the second it assigns a negative charge to each residue. Then, HyPare calculates how the changed charge affects the association rate.Lets take a look at chain B of 1jtg:

Using the mouse over feature we can get valuable information:

  1. The graph shows the analysis for each residue number of chain B. The Y value shows the change in the rate of association when a residue is mutated.
  2. The green points indicate the change in the rate when a residue is assinged a positive charge. If a residue is already positive, e.g. LYS, then a rate change of 1 is expected.
  3. The orange points indicate the change in the rate when a residue is assigned a negative charge.If a residue is already negative, e.g. GLU, then a rate change of 1 is expected.
  4. Points marked by an X are residues that appear at the interface, or binding site. Ideally, we are looking for points that increase the rate of association and that are not at the binding site.
  5. from the analysis we see that when ASP163 is assigned a positive charge (e.g. substituted with LYS), the rate increases by more than 12 fold. Luckily, ASP163 is not located at the interface, as can also be seen in the JMOL applet.
Jmol Applet:   

  1. Red colors indicate increased rate of association upon mutation.
  2. Blue colors indicate decreased rate of association upon mutation.
  3. Note that the coloring is in red/white/blue scale and it is relative to the complex in question.
JMOL is a JAVA version of Rasmol. Here are some tips for using the applet:
  • Left click with the mouse to rotate the molecule.
  • Hold down the shift button and click the mouse left button to zoom in/out.
  • hold down the shif button and double click the mouse left button to move the molecule.
  • Right click with your mouse to view more options.
  • Double click on one atom and then double clicking on another will show the distance between them.
  • Single click on any atom will show its properties (name, number,chain) in the browser status bar (lower left corner).
Export of HyPare files  

HyPare lets you export the source data of all the figures that you see in the output page. At the bottom of that page you will find this section:

  • You may download each file separately or click on download all to export all files in zip format.
  • click on Download Residue Analysis to save the source data of the graphs in text tab delimited format.
  • click on Download positive kon to save the JMOl positive coloring scheme in pdb format. This file includes all the green points of the graph mapped to the bfactor coulmn of the pdb file.
  • click on Download negative kon to save the JMOl negative coloring scheme in pdb format. This file includes all the orange points of the graph mapped to the bfactor coulmn of the pdb file.
  • click on Download summary table to save head table that appears in the output in text format.
  • click on Download log file to save the errors file. This option is visible only if HyPare detected errors in the processing.

Need more information? please visit HyPare homepage or contact Yossi Shaul