Examples

CAVER Analyst examples

The following example demonstrates the usage of CAVER Analyst on a case study described in detail by Pavlova et al. More examples and information can be found in the supplementary material for the CAVER Analyst publication in the Bioinformatics journal. The supplement is available here.

Case study – Engineering enzyme activity

Redesigning access tunnels of the haloalkane dehalogenase DhaA resulted in 32-fold increase in activity with toxic environmental pollutant 1,2,3-trichloropropane (TCP). The study demonstrates the power of combining rational design with directed evolution focused to the access tunnels. Additional information can be found in the article by Pavlova et al. published in Nature Chemical Biology.

1. Loading the structures
The study was performed with wild type and C176Y mutant of the haloalkane dehalogenase DhaA. The users are provided with the structures in PDB format wt_dhaa.pdb and m1_dhaa.pdb. These structures can be obtained by downloading examples.zip and unzipping the 'structures' folder.


2. Setting the binding site for tunnel computation
The CSA database does not contain any records for these structures thus the user must define the starting point manually, e.g., by defining the surrounding residues. In both structures, the active site is defined by the residues 106, 107, 130, and 272. They can be set using the 'Add Item...' button in the Tunnel Computation window, section Surrounding items. It opens the following window:




3. Launching tunnel computation
In this case the parameters present in the Tunnel Computation window or the CAVER 3 Computation Settings should have the following values:
• Maximum distance (Å)= 5
• Minimal probe radius = 0.7
The computation is then launched using the 'Compute Tunnels' button. This is performed for both structures and then their structure alignment can be achieved using Structure → Alignment.


4. Interpretation of results
The active site of wild type DhaA is an occluded cavity with two major access tunnels: (i) the main tunnel and (ii) the slot tunnel (Figure 1). Mutant carrying substitution in the main tunnel (C176Y) limits the access of water molecules to the active site cavity and resulting in higher activity with TCP (see Figure 2).





Figure 1.The main (in blue) and the slot (yellow) tunnels of the haloalkane dehalogenase DhaA.



Figure 2.Comparison of the wild type DhaA (left) and the DhaA mutant C176Y (right). The main tunnel is closed by the introduced mutation (in orange).