General preparation.

Make database file for the modified residues.

For plastocyanin, I will define two new types of residues: HIC, which will be a histidine coupled to a copper ion, and which will take the place of HIS 37 in the "real" sequence; and MEM, which is a modified methionine where the sulfur atom is of type "SM" rather than type "S". "SM" is a type I made up, and will use to create special force field parameters for MET 92, which is bonded to the copper ion with a fairly long bond.

(Note that you might want to use Antechamber for similar projects, but that Antechamber only works on complete molecules, not on fragments of the type considered here.)

Here are the input files for these two residues: 

+-------------------------------------------------------------------------+
|				 hicu.in				  |
+-------------------------------------------------------------------------+
|    0	  0    2							  |
|									  |
|HISTIDINE PLUS 							  |
|hicu.db4								  |
| HIC  INT     1							  |
| CORR OMIT DU	 BEG							  |
|   0.00000								  |
|   1  DUMM  DU    M	0  -1  -2     0.000	0.000	  0.000   0.00000 |
|   2  DUMM  DU    M	1   0  -1     1.449	0.000	  0.000   0.00000 |
|   3  DUMM  DU    M	2   1	0     1.522   111.100	  0.000   0.00000 |
|   4  N     N	   M	3   2	1     1.335   116.600	180.000  -0.34790 |
|   5  H     H	   E	4   3	2     1.010   119.800	  0.000   0.27470 |
|   6  CA    CT    M	4   3	2     1.449   121.900	180.000  -0.13540 |
|   7  HA    H1    E	6   4	3     1.090   109.500	300.000   0.12120 |
|   8  CB    CT    3	6   4	3     1.525   111.100	 60.000  -0.04140 |
|   9  HB2   HC    E	8   6	4     1.090   109.500	300.000   0.08100 |
|  10  HB3   HC    E	8   6	4     1.090   109.500	 60.000   0.08100 |
|  11  CG    CC    S	8   6	4     1.510   115.000	180.000  -0.00120 |
|  12  ND1   NB    B   11   8	6     1.390   122.000	180.000  -0.15130 |
|  13  CU    CU    E   12  11	8     2.050   126.000	  0.000   0.38660 |
|  14  CE1   CR    B   12  11	8     1.320   108.000	180.000  -0.01700 |
|  15  HE1   H5    E   14  12  11     1.090   120.000	180.000   0.26810 |
|  16  NE2   NA    B   14  12  11     1.310   109.000	  0.000  -0.17180 |
|  17  HE2   H	   E   16  14  12     1.010   125.000	180.000   0.39110 |
|  18  CD2   CW    S   16  14  12     1.360   110.000	  0.000  -0.11410 |
|  19  HD2   H4    E   18  16  14     1.090   120.000	180.000   0.23170 |
|  20  C     C	   M	6   4	3     1.522   111.100	180.000   0.73410 |
|  21  O     O	   E   20   6	4     1.229   120.500	  0.000  -0.58940 |
|									  |
|LOOP									  |
| CG   CD2								  |
|									  |
|IMPROPER								  |
| -M   CA   N	 H							  |
| CA   +M   C	 O							  |
| CE1  CD2  NE2  HE2							  |
| CG   NE2  CD2  HD2							  |
| ND1  NE2  CE1  HE1							  |
| ND1  CD2  CG	 CB							  |
|									  |
|DONE									  |
|STOP									  |
+-------------------------------------------------------------------------+

+-------------------------------------------------------------------------+
|				  mem.in				  |
+-------------------------------------------------------------------------+
|    0	  0    2							  |
|									  |
|METHIONINE, with SM atom type for the sulfur				  |
|mem.db4								  |
| MEM  INT     1							  |
| CORR OMIT DU	 BEG							  |
|   0.00000								  |
|   1  DUMM  DU    M	0  -1  -2     0.000	0.000	  0.000   0.00000 |
|   2  DUMM  DU    M	1   0  -1     1.449	0.000	  0.000   0.00000 |
|   3  DUMM  DU    M	2   1	0     1.522   111.100	  0.000   0.00000 |
|   4  N     N	   M	3   2	1     1.335   116.600	180.000  -0.41570 |
|   5  H     H	   E	4   3	2     1.010   119.800	  0.000   0.27190 |
|   6  CA    CT    M	4   3	2     1.449   121.900	180.000  -0.02370 |
|   7  HA    H1    E	6   4	3     1.090   109.500	300.000   0.08800 |
|   8  CB    CT    3	6   4	3     1.525   111.100	 60.000   0.03420 |
|   9  HB2   HC    E	8   6	4     1.090   109.500	300.000   0.02410 |
|  10  HB3   HC    E	8   6	4     1.090   109.500	 60.000   0.02410 |
|  11  CG    CT    3	8   6	4     1.525   109.470	180.000   0.00180 |
|  12  HG2   H1    E   11   8	6     1.090   109.500	300.000   0.04400 |
|  13  HG3   H1    E   11   8	6     1.090   109.500	 60.000   0.04400 |
|  14  SD    SM    S   11   8	6     1.810   110.000	180.000  -0.27370 |
|  15  CE    CT    3   14  11	8     1.780   100.000	180.000  -0.05360 |
|  16  HE1   H1    E   15  14  11     1.090   109.500	 60.000   0.06840 |
|  17  HE2   H1    E   15  14  11     1.090   109.500	180.000   0.06840 |
|  18  HE3   H1    E   15  14  11     1.090   109.500	300.000   0.06840 |
|  19  C     C	   M	6   4	3     1.522   111.100	180.000   0.59730 |
|  20  O     O	   E   19   6	4     1.229   120.500	  0.000  -0.56790 |
|									  |
|IMPROPER								  |
| -M   CA   N	 H							  |
| CA   +M   C	 O							  |
|									  |
|DONE									  |
|STOP									  |
+-------------------------------------------------------------------------+

I made mem.in just by copying the relevant portions of the methionine entry from all_amino94.in in the database directory, changing the atom type of the sulfur, and adding appropriate first and last lines. Similar things were done for the histidine residue (starting from the library's HIP residue), except that I added a copper atom bonded to ND1.

Do some editing of the PDB file.

Several small changes need to be made to the input PDB file to make it work with AMBER:

  1. First, we need to split of the HOH water residues into a separate file, say watpdb, and remove them from the main PDB file (call this modified file 1plc.nowat.pdb). Further, the remarks in this pdb file indicate that waters #183 and #187 are a disordered pair, and should not both be present. So, I arbitrarily choose to delete #187 and to keep #183. [Note that AMBER by default will also choose only the principal position for disordered side chains, i.e. the "A" conformation if there is more than one. But this is done automatically, and does not require editing. If you want to start a simulation from the "B" conformation of a side chain, you need to manually edit the PDB file to remove the "A" conformation and blank-out the alternate conformation flag for the atoms you want AMBER to use.] For some reason, these two disordered waters were both put in the PDB file, and not assigned as alternate conformers. Generally speaking, you have to look carefully at a PDB file before really using it.

    An alternative is to simply strip out the "crystallographic" waters and not use them at all. This is most appropriate if you are planning an MD or free energy simulation that will go through an extensive equilibration period before the "real" simulation begins. One goal of equilibration is to minimize dependence upon the starting conditions, and certainly the individual water molecules will move around a lot during any decent equilibration. At that point, the fact that you went to some trouble to originally place the waters in some nice positions may be irrelevant. Or maybe not; opinions differ on this matter, which is why we try to provide flexible tools in AMBER. For this tutorial, we will not use the "crystallographic" waters in our starting structure.

    One final change involves residue names. PDB files do no distinguish between cysteine residue that are involved in bonds to other things (and hence which have no proton on the sulfur atom) and "free" residues that do have such a proton. Molecular mechanics studies need to make this sort of distinction. Since residue 84 in plastocyanin has the sulfur atom bonded to the copper ion, I changed its name from CYS to CYX. Similar comments apply to histidines: molecular mechanics studies need to know (or guess, or assume) whether the histidine has a proton bonded at the `delta` position (HID), at the `epsilon` position (HIE), or at both (HIP). This is pretty easy for plastocyanin, since the two histidine side chains are both bound to copper through the ND1 nitrogen. So we initially change both HIS residues to HIE, in order to tell AMBER to put the protons on the NE2 nitrogen. (Note that in many other proteins, it will often be reasonable to assign surface-accessible histidines to be protonated, residue name HIP.)

  2. Next, we need to work on the proton names in the main protein file. Most PDB crystallographic files do not have protons, so the protonate program is used to add them. Even here, we want to change the names to NMR conventions as described above, so we will still use protonate . This program also does sanity and chirality checking, so it is generally a good idea to use it prior to putting any PDB file into AMBER. Now run:

    csh:
    ( protonate -k -d PROTON_INFO < 1plc.nowat.pdb > 1plc.nowat.H.pdb ) >& protonate.out

    sh:
    protonate -k -d PROTON_INFO -i 1plc.nowat.pdb -o 1plc.nowat.H.pdb -l protonate.out

    The -k option changes the names but "keeps" the positions of the protons in the input pdb file.   The -d  option specifies the file (PROTON_INFO) containing information on how to build and name protons.  As in other examples, you need to use the location of the PROTON_INFO file on your machine in place the location listed above.

  3. Next, I moved the copper ATOM card from the end of the pdb file into residue 37, changing its residue name to "HIC" and its residue number to 37. I also changed the residue name for the rest of atoms of residue 37 from "HIE" to "HIC", and changed the residue name for residue 92 from "MET" to "MEM" as described above. I call this file 1plc.protein.pdb .

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