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Multi-Probe Characterization


 1.  Goal

 2.  Introduction

 3.  Input

 4.  Chain and heteroatom selection

 5.  Box selection

 6.  Probe and algorithm selection

 7.  Runtime estimation

 8.  Output visualization

 9.  Output interpretation

10.  Reference

1.  GOAL

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- To facilitate comparison and combination of MIF clusters calculated with different probes, clustering algorithms and energy cutoffs;

- To allow identification of subsites with different molecular interaction properties in a larger binding site.


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  SiteHound-web, which was also developed by Sanchez Lab, calculates the molecular interaction field (MIF) of a protein with one of four chemical probes and one of two clustering algoritms, using a single default energy cutoff. Multi-Probe Characterization in SiteComp provides a platform to compare the MIF clusters detected with different parameters, and especially to combine MIF clusters detected with different probes.

  Adenylate kinase facilitates the transfer of a phosphoryl group from ATP to AMP. PDB entry 1aky is the structure of yeast adenylate kinase ligated with inhibitor AP5P and IMD. Occupying both the ATP site and the AMP site, AP5P mimicks the structure of an ATP molecule coupled to an AMP molecule. AP5P is negatively charged in the middle, which is the region of phosphate transfer, and hydrophobic on both ends due to the adenosine groups. [1]

  This tutorial illustrates how SiteComp could help identifying subsites with different molecular interaction properties in the active site of adenylate kinase.

  The example output page used in this tutorial can be found here.


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On the input page:

(1) Set task as "analyze one protein ".

(2) Enter a protein PDB code or upload a PDB file. For example, enter "1aky ".

(3) Click on "Submit ".

Note: Alternatively, one could start by using SiteHound-web to identify binding sites of one protein, or by using SiteComp's Binding site comparison option to identify subsites showing different ligand-binding properties. Either way, there will be buttons on the output page for single cluster analysis, which links to the Box Selection step with a default box enclosing the indicated region of interest.


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(1) If there are more than 1 chain in the protein, select chains for calculation.

(2) If there are heteroatoms, select the ones you intend to display on the output page. In this example, select AP5P.

(3) Click on "Submit ".

Note: This step is skipped if the protein contains only 1 chain and no heteroatoms. Unselected chains and selected heteroatoms could be displayed at later steps only for the purpose of visual reference, but they will be excluded for calculation.


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Define a calculation box within which SiteComp analysis will be performed. To ensure informative results, use a relatively small box enclosing the region of interest. In this example, this is the binding site of AP5P. It is recommended that a margin of a few angstroms is left in each dimension.

(1) For better visualization, use simple mouse actions and buttons in Section 1 to manipulate the molecule in Jmol applet.

(2) Box center could be set in Section 2 in three ways: 1. clicking on an atom in Jmol applet; 2. specifying a residue number; 3. specifying center coordinates. Box center will be moved to the specified atom/residue/coordiantes.

(3) Box dimensions could be set in Section 3 (Unit: angstrom).

(4) Ready, click on "Multi-probe characterization " in Section 4.

Initial view:

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Manipulated View (obtained with the following steps: 1. hiding protein backbones; 2. showing and labeling AP5P; 3. moving box to <10.000, 26.000, 17.000>; 4. resizing box to 17*26*18 to encompass AP5P):

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(1) Select probes for EasyMIFs calculation. For example, to compare the MIF clusters detected with a hydrophobic probe and a negatively charged probe, select CMET and OP.

(2) Select clustering algorithms for SiteHound. For example, select Average Linkage.

(3) To set energy cutoffs, select "Advanced Options ". Otherwise, the default cutoffs will be used.

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(4) Click on " >> " to start calculation.

Note: Please refer to EASYMIFs & SITEHOUND User's Guide for details about the different probes and clustering algoritms.


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Upon submission, SiteComp provides a runtime estimation and a link to the output page. You will be redirected to the output page when calculation finishes (usually within a few minutes). The output page will be kept on the server for 30 days, during which period it can be revisited as many times as desired.


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(1) For better visualization, use simple mouse actions and Section 1 to manipulate the molecules in Jmol applet. The size of Jmol applet could also be altered.

(2) Use Section 2 to visualize each set of clusters. Available options include:
  - visualize the clusters calculated with different energy cutoffs.
  - visualize a subset of clusters. By default, only the first cluster (the one with the most favorable energy value) is displayed. You may choose to display up to 10 clusters.
  - visualize and label the sidechains surrounding the clusters currently being displayed.
  - change the color of clusters.
  - color cluster points according to absolute energy for better visualization of energy distribution. The points with lightest and darkest colors are the ones with least and most favorable energy values from all clusters resulting from each combination of probe, clustering algorithm and energy cutoff.

Initial View:

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(3) To download the output files, click on "DOWNLOAD " in Section 1.

* Explanation of files in the downloaded package   

Note: The .pdb files can be used to display the proteins, heteroatoms and clusters in molecular visualization softwares such as PyMol or Jmol. Please refer to EASYMIFs & SITEHOUND User's Guide for detailed description of each file type.

(4) To compare a different set of probes, clustering algorithms or energy cutoffs, click on " << ".


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   As shown in Manipulated View 1, the first two clusters detected with the CMET probe, using energy cutoff e=-10.0 (shown in dark red) describe the subsites for the two adenosine groups.

Manipulated View 1 (obtained with the following steps: 1. hiding protein backbones, showing AP5P; 2. hiding clusters detected by the "OP" probe; 3. displaying top two clusters detected by the "CMET" probe, e=-10.0; 4. coloring cluster points by energy; 5. rotating the molecule by dragging in Jmol applet):

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   As shown in Manipulated View 2, the first cluster detected with the OP probe, using energy cutoff e=-20.0 (shown in green) describes the subsite for the phosphate groups. There are several positively charged residues, including arginine and lysine, surrounding this cluster, and thus create a favorable environment for the negatively charged phosphoryl groups. The second and third clusters in this group(not shown) are close to the adenosine groups, but the signal is much weaker as compared to the CMET probe results.

Manipulated View 2 (starting with Manipulated View 1, obtained with the following steps: 1. hiding clusters detected by the "CMET" probe; 2. displaying clusters detected by the "OP" probe; 3. selecting energy cutoff e=-20.0 for the OP probe):

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   Comparison of the results with the CMET and OP probes illustrates the different properties of the two probes. The CMET probe is hydrophobic and therefore detects binding sites regardless of electrostatic charge, while the OP probe is negatively charged, and therefore is more sensitive to positively charged subsites. Actually, by combining the results with CMET and OP probes, SiteComp provides information about the whole AP5P binding site (Manipulated View 3).

Manipulated View 3 (starting with Manipulated View 1, obtained with the following steps: 1. displaying clusters detected by the "OP" probe; 2. selecting energy cutoff e=-20.0 for the OP probe):

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Abele, U. and Schulz, G.E. (1995) High-resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer, Protein Sci, 4, 1262-1271.

For help and questions, please email sitecomp@sanchezlab.org