S scaled by a factor of 0.01. In order to be compatible with the energy score optimization algorithm, the energy values have to be P pairwise decomposable, i.e. of the form P Etotal i Ei z i,j Ei,j . Ei are the self energies of the variables(side chain conformers or ligand poses), i.e. their inherent energies and the energies with the fixed protein parts, and Ei,j the pairwise energies between the variables. As we are interested in improving Bromopyruvic acid custom synthesis binding affinity, we chose to upscale the binding energies by a factor of ten for CADDSuite scores and a factor of 100 for Autodock Vina scores to arrive at absolute values that are in the same range as the AMBER packing energies. The Ei and Ei,j energy tables are computed for all side chain conformers at the pocket positions and the ligand poses. The problem of finding the minimum energy conformation is formulated in graph-theroretic terms [32] and solved using the MPLP algorithm by Sontag et al. [33]. The energy minimum identifies the best design with corresponding score values and conformation. POCKETOPTIMIZER is realized as a collection of binaries and scripts that perform the different subtasks. It was developed and tested on Ubuntu Linux 10.04 operating system. AMBER packing energy calculations are implemented in C++ using BALL [41], so is the ligand pose generation tool. Protein-ligand energies for CADDSuite are calculated with a scorer binary implemented in C++ as well, vina energies are calculated using the vina binary 125-65-5 provided with the Autodock vina software distribution. The side chain conformer library contains the structures of the amino acid side chains in PDB and SDF formats. Several Python scripts are provided that interface between the different parts and allow a convenient conducting of a protein design task with the POCKETOPTIMIZER pipeline. Intermediate result are stored in standard file formats, SDF and PDB formats for structural data, and CSV files for energy tables. This allows the user to easily inspect this data with standard tools. It also facilitates the possibility to use a different approach for one of the modules, e.g. a different docking function, while the rest of the pipeline can remain unaltered.Setup for PocketOptimizer BenchmarkThe protein structures were briefly minimized using CHIMERA’s [46] AMBER implementation. Amino acids of the binding pocket positions that were allowed to change conformations in the ?calculations had to have a distance smaller than 4 A of at least one side chain atom to the ligand or to one of the residues that are mutable. Ligand conformers were rotated by 620u around each ?axis and translated by 0.5 A in each direction to create the ligand poses. If this resulted in more than 3000 poses, the conformers were filtered by similarity to the crystal structure conformation until meeting the max 3000 poses criterion. For proteins that contain metals in their binding pocket that are coordinated by the ligand, the ligand poses were filtered for poses that are geometrically compatible for coordination.Rosetta Design SetupThe ROSETTA enzyme design application as implemented in ROSETTA 3.3 [30] was used with parameters closely following the relevant documentation. Protein structures were briefly minimized using the ROSETTA receptor preparation application provided for this task, generating ten resulting structures of which the one with the best energy was used for the design runs. Ligand conformers were generated using OMEGA2, ligand charges added.S scaled by a factor of 0.01. In order to be compatible with the energy score optimization algorithm, the energy values have to be P pairwise decomposable, i.e. of the form P Etotal i Ei z i,j Ei,j . Ei are the self energies of the variables(side chain conformers or ligand poses), i.e. their inherent energies and the energies with the fixed protein parts, and Ei,j the pairwise energies between the variables. As we are interested in improving binding affinity, we chose to upscale the binding energies by a factor of ten for CADDSuite scores and a factor of 100 for Autodock Vina scores to arrive at absolute values that are in the same range as the AMBER packing energies. The Ei and Ei,j energy tables are computed for all side chain conformers at the pocket positions and the ligand poses. The problem of finding the minimum energy conformation is formulated in graph-theroretic terms [32] and solved using the MPLP algorithm by Sontag et al. [33]. The energy minimum identifies the best design with corresponding score values and conformation. POCKETOPTIMIZER is realized as a collection of binaries and scripts that perform the different subtasks. It was developed and tested on Ubuntu Linux 10.04 operating system. AMBER packing energy calculations are implemented in C++ using BALL [41], so is the ligand pose generation tool. Protein-ligand energies for CADDSuite are calculated with a scorer binary implemented in C++ as well, vina energies are calculated using the vina binary provided with the Autodock vina software distribution. The side chain conformer library contains the structures of the amino acid side chains in PDB and SDF formats. Several Python scripts are provided that interface between the different parts and allow a convenient conducting of a protein design task with the POCKETOPTIMIZER pipeline. Intermediate result are stored in standard file formats, SDF and PDB formats for structural data, and CSV files for energy tables. This allows the user to easily inspect this data with standard tools. It also facilitates the possibility to use a different approach for one of the modules, e.g. a different docking function, while the rest of the pipeline can remain unaltered.Setup for PocketOptimizer BenchmarkThe protein structures were briefly minimized using CHIMERA’s [46] AMBER implementation. Amino acids of the binding pocket positions that were allowed to change conformations in the ?calculations had to have a distance smaller than 4 A of at least one side chain atom to the ligand or to one of the residues that are mutable. Ligand conformers were rotated by 620u around each ?axis and translated by 0.5 A in each direction to create the ligand poses. If this resulted in more than 3000 poses, the conformers were filtered by similarity to the crystal structure conformation until meeting the max 3000 poses criterion. For proteins that contain metals in their binding pocket that are coordinated by the ligand, the ligand poses were filtered for poses that are geometrically compatible for coordination.Rosetta Design SetupThe ROSETTA enzyme design application as implemented in ROSETTA 3.3 [30] was used with parameters closely following the relevant documentation. Protein structures were briefly minimized using the ROSETTA receptor preparation application provided for this task, generating ten resulting structures of which the one with the best energy was used for the design runs. Ligand conformers were generated using OMEGA2, ligand charges added.
