(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)
Copyright Ross Walker 2006

TUTORIAL 4 - SECTION 1

Simulating a Solvated Protein that Contains Non-Standard Residues
(Simple Version)

By Ross Walker

Stage 1 - Do some editing of the PDB file.

The pdb file we will use is PDB ID: 1PLC - 1PLC.pdb.

You should always read all of the header information in a pdb file since it often contains important information about disordered pairs etc. Often 1 pdb file can also contain a series of different structures / inhibitors etc for the same protein system. In this pdb we see that waters #187 and #183 form a disordered pair and so both should not be present.

REMARK   4                                                              1PLC  83
REMARK   4 HOH 187 AND HOH 183 FORM A DISORDERED PAIR AND BOTH ARE NOT  1PLC  84
REMARK   4 PRESENT SIMULTANEOUSLY.                                      1PLC  85

Thus I  will arbitrarily remove water 187 from the pdb file and keep 183. I will also remove all of the connectivity data at the end of the PDB as this will not be used by XLeap. Each of our water molecules here is an individual residue. Normally we need to add TER cards between residues that are not bound together in a chain. Fortunately we don't need to do that for our crystallographic waters since XLeap is smart enough to know that each of our waters is a separate molecule. This is defined in the WAT unit. Note, if you were to use different solvents here, such as chloroform, it would be necessary to add the TER cards.

Since pdb files do not distinguish between cysteine residues that are involved in bonds or other things (and hence which have no proton on the sulphur atom) we need to edit the relevant cysteine residues to correct for this. The residue name used by Leap for regular protonated cysteine residues is CYS, for deprotonated (-ve charge) and/or bound to metal atoms it is CYM and for cysteine residues involved in disulphide bridges and other bonds it is CYX. Since cysteine 84 in plastocyanin is bonded to the copper ion we need to change the residue name of residue 84 from CYS to CYM. The same is true for histidine residues which can be protonated in the 'delta' position (HID), the epsilon position (HIE) or at both (HIP). Fortunately in plastocyanin this is fairly easy since there are only two histidine residues (37 and 87) both of which are bonded to the copper via the 'delta' nitrogen. Thus they must both be protonated on the epsilon nitrogen. We will thus change both histidine residue names (37 and 87) from HIS to HIE. Leap will then add the correct number of protons in the correct positions.

Here is the modified pdb file so far: 1PLC_mod.pdb

The next stage before we can use the PDB is to sort out the non-standard hydrogen names. While it is possible to use protonate to correct the non-standard hydrogen names, converting them from NMR conventions to PDB conventions, experience has shown that hydrogen positions in NMR structures can't always be trusted. The best option therefore is to remove all the protons and allow Leap to add them back in at standard positions. Since we will typically minimise our system before running MD this shouldn't pose any problems.

Here is the pdb with the protons removed: 1PLC_mod2.pdb

The next stage is to work out how we are going to treat the copper atom. For this example we shall keep it simple and just treat the copper as a +1 ion bound to the nearby residues. Ideally we would like to make the 4 copper bound residues new non-standard residues and then re-fit all the charges and parameters for these residues and the copper atom. This is a large amount of work, however, and well beyond the scope of this tutorial. For this reason we shall treat the copper atom as simply as possible. We will thus make the copper atom its own residue called CUA (note: in picking a residue name anything would do as long as it is 3 characters long and not currently in use... You can use the list command in Leap to check what residue names are currently being used.).

So we will edit our pdb file and change the copper residue name to CUA.

ATOM   1538  OD1 ASN    99       7.523  13.716  33.177  1.00 33.45      1PLC1661
ATOM   1539  ND2 ASN    99       8.763  14.800  34.732  1.00 31.51      1PLC1662
ATOM   1540  OXT ASN    99       8.932  10.327  32.908  1.00 32.10      1PLC1663
TER    1547      ASN    99                                              1PLC1670
HETATM 1548 CU   CUA   100       7.050  34.960  18.716  1.00  8.78      1PLC1671
HETATM 1549  O   HOH   101      17.504  16.825  14.073  1.00 20.28      1PLC1672
HETATM 1550  O   HOH   102      18.877  15.088  18.086  1.00 22.16      1PLC1673
HETATM 1551  O   HOH   103      11.165  21.823  31.513  1.00 16.99      1PLC1674

The original pdb file also contained several "alternate" configurations for some residues. E.g. for LYS 30 we had

ATOM    426  N   LYS    30      -0.930  27.774  20.957  1.00  8.07      1PLC 549
ATOM    427  CA  LYS    30      -2.028  28.602  20.421  1.00 10.86      1PLC 550
ATOM    428  C   LYS    30      -1.629  30.029  20.254  1.00  9.16      1PLC 551
ATOM    429  O   LYS    30      -1.226  30.665  21.243  1.00  7.63      1PLC 552
ATOM    430  CB ALYS    30      -3.201  28.489  21.415  0.50 13.41      1PLC 553
ATOM    431  CB BLYS    30      -3.250  28.517  21.354  0.50 15.09      1PLC 554
ATOM    432  CG ALYS    30      -4.397  29.366  21.249  0.50 16.84      1PLC 555
ATOM    433  CG BLYS    30      -4.600  28.495  20.646  0.50 21.50      1PLC 556
ATOM    434  CD ALYS    30      -5.681  28.891  21.893  0.50 20.64      1PLC 557
ATOM    435  CD BLYS    30      -5.745  28.171  21.589  0.50 24.43      1PLC 558
ATOM    436  CE ALYS    30      -5.527  28.212  23.225  0.50 23.18      1PLC 559
ATOM    437  CE BLYS    30      -5.585  26.973  22.460  0.50 24.88      1PLC 560
ATOM    438  NZ ALYS    30      -6.825  28.052  23.929  0.50 20.02      1PLC 561
ATOM    439  NZ BLYS    30      -5.971  25.681  21.860  0.50 26.52      1PLC 562
ATOM    440  H   LYS    30      -0.661  27.945  21.802  1.00  8.96      1PLC 563
ATOM    441  HA  LYS    30      -2.302  28.229  19.582  1.00 10.61      1PLC 564
ATOM    442 1HB ALYS    30      -3.380  27.572  21.665  0.50 14.09      1PLC 565
ATOM    443 1HB BLYS    30      -3.134  27.712  21.919  0.50 15.93      1PLC 566
ATOM    444 2HB ALYS    30      -2.700  28.880  22.297  0.50 13.96      1PLC 567
ATOM    445 2HB BLYS    30      -3.188  29.326  21.939  0.50 15.16      1PLC 568
ATOM    446 1HG ALYS    30      -4.210  30.316  21.598  0.50 18.44      1PLC 569
ATOM    447 1HG BLYS    30      -4.784  29.428  20.234  0.50 20.74      1PLC 570
ATOM    448 2HG ALYS    30      -4.609  29.538  20.265  0.50 17.98      1PLC 571
ATOM    449 2HG BLYS    30      -4.629  27.910  19.855  0.50 20.04      1PLC 572
ATOM    450 1HD ALYS    30      -6.278  29.712  22.058  0.50 21.47      1PLC 573
ATOM    451 1HD BLYS    30      -5.929  28.976  22.170  0.50 24.46      1PLC 574
ATOM    452 2HD ALYS    30      -6.224  28.346  21.263  0.50 21.94      1PLC 575
ATOM    453 2HD BLYS    30      -6.606  28.094  21.037  0.50 24.54      1PLC 576
ATOM    454 1HE ALYS    30      -5.138  27.298  23.130  0.50 22.69      1PLC 577
ATOM    455 1HE BLYS    30      -4.709  26.867  22.883  0.50 25.84      1PLC 578
ATOM    456 2HE ALYS    30      -4.956  28.735  23.845  0.50 23.28      1PLC 579
ATOM    457 2HE BLYS    30      -6.257  27.063  23.262  0.50 25.75      1PLC 580
ATOM    458 1HZ ALYS    30      -7.360  28.765  23.779  0.50 21.25      1PLC 581
ATOM    459 1HZ BLYS    30      -6.721  25.740  21.353  0.50 26.03      1PLC 582
ATOM    460 2HZ ALYS    30      -7.206  27.240  23.677  0.50 21.83      1PLC 583
ATOM    461 2HZ BLYS    30      -5.279  25.211  21.533  0.50 25.17      1PLC 584
ATOM    462 3HZ ALYS    30      -6.682  27.986  24.852  0.50 21.60      1PLC 585
ATOM    463 3HZ BLYS    30      -6.289  25.095  22.599  0.50 25.92      1PLC 586

By default Leap will use the A conformation and disregard the others. This is fine for our purposes here. If we specifically wanted to start out in one of the other confirmations we would need to remove the A conformation from the file.

The final thing we need to do is add a TER card between our copper atom and our first crystallographic water atom. We will be adding the bonds to the copper atom manually within Leap since it is not strictly part of the protein chain. Hence the TER cards either side of it act to stop Leap trying to make it part of the protein chain and getting upset.

Here is the pdb file after the modifications above: 1PLC_mod_final.pdb

CLICK HERE TO GO TO SECTION 2

(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)
Copyright Ross Walker 2006