Step 1. Generate some starting coordinates.
The first step is to obtain starting coordinates. We begin with the file 6pti.pdb, exactly as distributed by the Protein Data Bank and Brookhaven. This file (as with most Brookhaven files) needs some editing before it can be used by Amber. First, alternate conformations are provided for residue 50, so we need to figure out which one we want. For this example, we choose the "A" conformation, and manually edit the file to remove the alternate conformers. Second, coordinates are provided for a phosphate group and a variety of water molecules. These are not needed for the calculation we are pursuing here, so we also edit the file to remove these. Third, the cysteine residues are involved in disulfide bonds, and again need to have their residue names changed in an editor from CYS to CYX to reflect this. Let's call this modified file 6pti.mod.pdb. Finally, hydrogen positions are not included, so we run the Amber program protonate to provide these:
protonate -d $AMBERHOME/dat/PROTON_INFO < 6pti.mod.pdb > 6pti.H.pdb
In other situations, many different programs could be used to generate starting coordinates, but the basic ideas are the same: somehow generate what you want in a "pdb" format, then run the result through protonate. We recommend doing the last step even if protons are present, since protonate performs a number of checks on the correctness and naming of hydrogen atoms.
Step 2. Run LEaP to generate the parameter and topology file.
This is a fairly straightforward exercise in loading in the pdb file, adding the disulfide cross links, and saving the resulting files. Type the following command in either tleap or xleap:
Step 3. Perform some minimization.
Use this script:
+-------------------------------------------------------------+ | Running minimization for BPTI | +-------------------------------------------------------------+ |cat << eof > min.in | |# 200 steps of minimization, distance-dependent dielectric | | &cntrl | | maxcyc=200, imin=1, cut=12.0, igb=3, ntb=0, ntpr=10, | | &end | |eof | |sander -i min.in -o 6pti.min1.out -c prmcrd -r 6pti.min1.xyz | |/bin/rm min.in | +-------------------------------------------------------------+
This will perform minimization (imin) for 200 steps (maxcyc),
using a nonbonded cutoff of 12 `angstroms' (cut) and a distance-dependent
dielectric constant (igb),
and intermediate results will be printed
every 10 steps
(ntpr). Text output will go to file 6pti.min1.out, and the final
coordinates to file 6pti.min1.xyz.