Any line in the parameter file that begins with a # is treated as a comment, i.e., it will be ignored. The rest of the lines are assumed to contain parameter values. The order of the parameters is not important, but there should only be one parameter per line. Also, extraneous parameters will be ignored. This allows the same parameter file to be used in all the programs that require a parameter file. It is worth using only one parameter file because many of the parameters are used by more than one program.
The first two parameters are
| DENSITY_MAP | ZZZ.map |
| DENSITY_GRID | ZZZ.grd |
The file specified by the parameter DENSITY_MAP is the XPLOR formatted electron density map file. The file specified by the parameter DENSITY_GRID is the output electron density grid file of Map2Grid and is an input file for Blob.
The next parameters you should chose based the portion of your electron density that you are interested in investigating. Basically, these parameters define the volume of density of interest. For example suppose you want to search a box bounded by
7 < Y < 42
-4 < Z < 29
with a .5 Å grid spacing then the parameters describing the grid should be
| XLO | 0.0 |
| YLO | 7.0 |
| ZLO | -4.0 |
| SPACING | 0.5 |
| X_GRID_PTS | 50 |
| Y_GRID_PTS | 70 |
| Z_GRID_PTS | 66 |
All length units are in Å. Keep in mind that you should allow enough of a cushion around the interesting density so that the molecule has room to move while not making the box too large (which is my way of saying you might have to fiddle with the grid size a little). If the search box extends beyond the area covered by the map, Map2Grid gives grid points outside the map a value of 0.0.
The next set of parameters control the simulated annealing protocol of Blob:
| N_RUNS | 100 |
| N_CYCLES | 20 |
| N_ACC_MAX | 3000 |
| N_REJ_MAX | 3000 |
| RT_0 | 50.0 |
| RT_FAC | .85 |
| STEP_SIZE_0 | 0.1 |
| STEP_SIZE_FAC | .95 |
| TAKE_MIN | 1 |
The values in the these parameters are the ones I have typically used for small flexible ligands but their values are largely left up to the user. The most important one is N_RUNS which is the number of final positions that will end up in the output file. The manner in which these parameters are used is described in the postscript version of the manual.
The next parameters describe the file that holds the molecule to be positioned:
| LIGAND | ZZZ.pdb |
| INPUT_FORMAT | 4 |
| OUTPUT_FORMAT | 10 |
The file specified by the parameter LIGAND is the file that you painstakingly prepared as described in section 2. The INPUT_FORMAT and OUTPUT_FORMAT specify the input format and the output format for the atom records. These are important to get right as they can increase the amount of information you extract from a set of positioning runs. The general subject of atom records is taken up in some detail in section 8.
Up to this point all the parameters are required. The rest of the parameters are optional depending on your situation.
The following parameters determine the manner in which Blob will calculate the score.
| INT_ENERGY_WT | 1.0 |
| IENERGY_PAR | ienergy.par |
| PROTEIN_WEIGHT | 1.0 |
The first two of these parameters determine how Blob scores the internal energy of the molecule. The INT_ENERGY_WT is the weight of the internal energy of the molecule relative to the density score for the molecule. Large values for this parameter increase the importance of the internal energy. The file specified by the IENERGY_PAR parameter should contain standard 12-6 Van der Waals parameters. The calculation of the internal energy is described in the postscript version of this manual. Thus the internal energy merely prevents internal steric clashes. If the parameter INT_ENERGY_WT is set to 0.0 or left out entirely Blob will not calculate an internal energy. The parameter PROTEIN_WEIGHT specifies how strongly to weight the protein-ligand interactions relative to the electron density score. If PROTEIN_WEIGHT is set to 0 or left out, Blob will not calculate the protein-ligand interaction score.
Finally, if you are positioning in the binding site of a protein, GridsRUs can be used to create the necessary grid files. If you wish to use GridsRUs to create electrostatic and atomic affinity grids then the following parameters are required (otherwise omit these parameters):
| PROTEIN_PDB | protein.pdb |
| PROTEIN_FORMAT | 4 |
| VDW_ENERGY_PAR | vdw.par |
| ATOM_TYPES | 30 |
The parameter PROTEIN_PDB specifies the file that contains the protein in whose binding site the ligand will be positioned. Note this file should be a typical pdb formatted file except that if you want the electrostatics included in the score you have to add hydrogens and charges to the protein using whatever program you like. Again the charge should be in the column after the bfactor column.
The parameter PROTEIN_FORMAT specifies the format of each of the ATOM records in the PROTEIN_PDB file. As a warning pay special attention to whether the chain letter is or is not there. The ATOM record format is described in section 8.
The parameter VDW_ENERGY_PAR specifies the file that contains Van der Waals parameters that GridsRUs is to use to calculate the Van der Waals grids. This file is formatted exactly as the IENERGY_PAR file with the only difference that the VDW_ENERGY_PAR need not be 12-6 parameters.
The ATOM_TYPES parameter tells GridsRUs which atomic affinity grids to make and tells Blob which atoms types to expect. The value of this parameter is determined from Table 13. Note that if you are not using the force field as part of the score this parameter should be set to 0 regardless of the atom types in the molecule you are positioning.
| Atom Type | Atom Num | Atom Flag |
| H | 0 | 1 |
| C | 1 | 2 |
| O | 2 | 4 |
| N | 3 | 8 |
| S | 4 | 16 |
| P | 5 | 32 |
| I | 6 | 64 |
| F | 7 | 128 |
| CL | 8 | 256 |
| BR | 9 | 512 |
| D | 10 | 1024 |
As an example, suppose your the ligand has only atom types C(2), O(4), N(8), and S(16).Thus ATOM_TYPES is set to 2+4+8+16=30. The type D might seem a bit odd. It is treated as a hydrogen but is intended to be used to better describe a hydrogen bond. Giving type D a shorter Van der Waals radius than type H allows the hydrogen bonds to be a more typical length. To use this trick both the Van der Waals radius of D must be set to something around .6 Å(rather than around 1.0 Åfor an H) and all hydrogens that can be donated must be labeled with a D rather than an H. This is roughly the approach used by AMBER [2]. Currently, GridsRUs and Blob recognize only these 10 atom types. I hope these 10 types are enough.
As a general warning It is actually kind of ugly when any of these programs gets a parameter file that is missing something. Do not be surprised the first time it happens. If you get an extremely long error message after invoking a command most likely your parameter file is missing something. Go to the beginning of the error message and it should say what is missing after which it lists every possible parameter it can be given. Knowing this can actually help. If you are not sure of a parameter name just guess. If you are wrong the program will tell you. If you are right all the better. Also, any extraneous parameters are just ignored. Thus the same parameter file can be used for all three programs.