Team:Virginia/Software

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← Modeling

MUT

Purpose

In order to generate and test a large list of mutants we build a software pipeline in Linux called MUT. MUT utilizes PyRosetta, Autodock Vina, and FoldX to mutate, test, and rank protein structures based on docking scores. This program was used to query for the optimal mutant structure for Cbz-Leu docking. The following is a visualization of our mutant synthetase with optimal structure.

Structure

Upon initiation, MUT asks for four main inputs: the protein pdb file that will be analysed, the residues that the program will mutate, the coordinates of the binding pocket for docking simulations, and a ligand pdb file. MUT first performs stability testing and docking to get baseline values for the future tests before it mutates the initial PDB file. At each iteration the residue is mutated to 17 other amino acids, not including Cys, Pro (as they induce kinks and are difficult to model) or itself. After mutagenesis is complete files undergo stability testing and docking to determine if the new mutant is stable and a better match for the ligand. Files that fail the tests go to the kennels. Below is a flowchart that explains the algorithm of the program.

The kennel system allows for two levels of simulation. The first and fastest way is the reductionist approach. This method disregards all files that fail any test at any point. This method is quick, but because of the variability of docking simulations, it is likely to miss key files. The exhaustive approach takes into consideration all possible mutations. After the reductionist approach is complete, all files are mutated to four mutants and re-scored. This method ensures that all possible combinations of mutations is tested, but is computationally intense.

Output

After a run is complete, all files are automatically ranked and ordered by binding values (more negative is better). Once an initial ranking has be completed, the user has the option to rerun docking on the selected mutants to confirm the simulation. Should the results look bad, mutants from a lower ranking can be selected or new residues can be picked.

Summary

Virginia iGEM designed a Linux-based protein engineering tool called MUT. This program is used to screen protein-ligand docking across possible protein mutants. It is written from a Centos shell, but is designed to be portable across distributions. MUT generates mutant structures, subjects the resultant proteins to stability testing, and ranks the results based on protein-ligand docking data. Three programs are used to accomplish this task: PyRosetta conducts computational mutagenesis, FoldX tests protein stability, and AutoDock Vina performs protein-ligand docking. All three of these programs are available for free with an educational license, and MUT is hosted on an open source site. MUT is designed to be configurable to any iGEM team’s needs. The user inputs to MUT a protein’s PDB file, residues of interest for mutation, a binding pocket, and a ligand. After the simulation runs, the program outputs a top ten list of mutant structures along with their files.

Source
MUT and other code