Example: DHFR + Methotrexate

Here, we briefly introduce how to analyze protein-ligand interactions with GIANT. As an example, we focus on dihydrofolate reductase (DHFR) complexed with an inhibitor, methotorexate (PDB-ID:3dfr).

1. Load the complex structure

To begin with, a structure of DHFR-methotorexate complex is loaded.

Go to "Complex Analyzer" by clicking the upper-right part of the entrance page.

A query complex can be specified in one of the two ways:

For this tutorial, we focus on DHFR-methotorexate complex, PDB-ID: 3dfr. Because this complex is deposited in our database, you can load it in the former way (a). Input the "3dfr" in the text box (and do NOT enter or click "load" button at this time).

Even if our database does not have the complex which you want to analyze, you can analyze it by uploading a .pdb file and specifying 3-letter code of a ligand.

After a moment, list of suggested complexes will be shown.

In GIANT, a complexes is identified by a combination of PDB-ID and 3-letter code of the ligand (and they are concatenated by underscore '_') e.g., "3dfr_MTX").

Choose the "3dfr_MTX" and click "load" button for analyzing the complex.

Recognition of amino groups in the pteridine

The structure of ligand, methotorexate, will appear in Jmol Java applet at left part of the window. You can rotate the view with dragging in the applet window.

In the right part, there are three tables showing list of amino acid residues, ligand atoms, and interactions.

Here, we'll try to see recognition of 2-amino group in the pteridine ring. The nitrogen atom is labelled as the number "3" in the Jmol applet (pink circle at left-bottom of the figure).

To analyze interactions of this atom, click the check box of the ligand atom-ID "3", in the ligand atom table.

Then, three amino acid residues, Ala6, Thr116, and Asp26, will appear. Orange lines between atoms indicate interacions with patterns.

The original paper reporting this X-ray structure (Bolin et al., J. Biol. Chem., 1982) described the interactions of these three amino acids. According to it, interactions between Thr116 and 2-amino group is conducted through a water molecule. Because GIANT does not have data of interactions with water molecules, GIANT shows the interaction as a direct contact.

But, you can confirms that this interaction is not so common; GIANT shows this interaction pattern was observed only five protein families.

To see more details of the interaction patterns between Asp26 and 2-amino group, see the interaction table and seek the rows with Residue Type "ASP", Residue ID "26", and Ligand ID "3".

Tables can be sorted by clicking the column headings.

Four interactions (rows) will be found for Asp26-atom3 interactions. One line between O and N in the 3-D view is belongs two interaction: interaction of the frgment Od-Cg-Cb, and that of Od-Cg-Od. Here, we will analyze one of them; click the interaction ID: 18713.

Spatial distribution of interaction patterns

Pattern Viewer shows spatial distributions of interaction patterns. Visibility of each Gaussian component, or interaction pattern, can be manipurated by the check box in the right table.

The table shows basic information about each interaction patterns:
IDID number for the interaction pattern
PiProbability of the interaction pattern in the Gaussian mixture model.
FamilyNumber of protein families using the interaction pattern in our dataset.
FreqNumber of complexes using the interaction pattern in our dataset.
CmpxLink to the list of protein-ligand complexes using the interaction pattern.