Hello Sindrila,
Adding residues to a protein is not an easy problem in general, and particularly for a protein missing 42 residues in the middle of the chain. You need to model the generation of a loop in each residue. Perhaps the most suitable program to do this is modeller - it has only a command line interface. I can tell you a bit about what you can do within leap for this problem. It may be soluble in leap, but it is tedious work. First, put a TER card in the gap and before you load the pdb in leap give the command clearpdbresmap. This causes the empty valences to be left alone so no atoms are added anywhere upon loading. Second, you can generate the missing peptide with the sequence command:
pep = sequence {res1 res2 res3 ......res42}
This peptide will automatically have a head nitrogen atom: pep.1.1; and a tail carbon atom pep.42.n, where n depends on what amino acid it is.
Now load your protein (but first remove all water molecules and any ligands - leave only the pure protein residues in the working pdb file - you can add the ligands back later by cutting & pasting).
mol=loadpdb protein.pdb
Now comes the first really important step. You must add the pep sequence to the first half of your molecule in the gap. Your last residue before the gap is mol.185 (according to what you say below)
Thus, you must make the peptide carbon (C') the tail of mol. Use the desc command to find out
Which atom number that is:
desc mol.185
If it is a glu, then the carbon is number 14. Let's assume that's what it is for definiteness.
set mol tail mol.185.14
Now join the peptide at this point using the sequence command, to get a new protein called pro:
pro = sequence {mol pep}
[Note: if you were to join it to the other end (mol.228), then leap changes the coordinates of all residues in the loaded protein after that and you have messed up your pdb file - since this is a tetramer, you can't allow that because you must maintain proper relations to the other chains.]
Now save the pdb file
savepdb pro protein.s1.pdb
Where s1 means step 1. You can add the same peptide to the other chains the same way, paying careful attention to the numbering of the residues so you add it in the right place. Use the desc command to make sure you are in the right place. It may be better to put each chain in a separate pdb file and work on each chain one by one. You can do the above in tleap, but the second
Important step requires that you use xleap. The task is to twist the added peptide so that the other end will be close to the mol.228 residue so you can make a bond between the tail and head atoms of the pair. So if you were using tleap, quit and start xleap. Load using the clearpdbresmap as before, call it mol. Now type
edit mol
This brings up the graphical editing window. It is kind of cryptic. You need to find the peptide that you added by zooming, rotating and moving your protein - in particular you want to find the end that is free, corresponding to pep.42. Hopefully you will also be able to see the N atom of mol.229. I imagine that the pep you added is nearly straight so it's sticking out horribly in some direction. You First want to click on the "select" tab and then click on the beginning part of the peptide and the end of it. The peptide will turn pink - you have now selected it. Now select the tab "twist". Using your mouse, fold this peptide as best you can to make the end of it come close to the target N atom. When and if you manage this, then generate a bond between the tail of pep.42 (C') and the head of mol.228 (N) using the makebond command. Now save your pdb file.
Note: it may be better to pre-fold your pep in xleap before you add it to the protein so that it is not a long straight peptide. This requires a lot of trial and error!
The loop you have generated needs to be energy minimized. It may have a horrible starting structure because you folded it by hand. You can choose to energy minimize the loop on a single chain and then collect all the chains together at the end in the same pdb file. The energy minimization may not give you a structure that you can completely trust for such a long added peptide. If you decide to go this route, write to me again and I will give you a protocol for energy minimization. It will take you awhile to just add the peptide without messing up the coordinates of the rest of your protein, and join it completely on both ends.
OK, this gives you a picture of the tedious process one needs to do within leap. For a peptide with 42 residues, I really recommend that you get the modeller program and use it instead. The program is free (type protein modeller in google), and requires some effort to learn to use as well.
I am doing this for a series of proteins myself, but use leap only for the cases where the added peptides are small (less than 10 residues). When missing residues occur at the beginning of the protein, then there is a problem because you can't add a peptide w/o changing the coordinates of the rest of the residues. This is OK if you only have one chain, but not for a multimer. This problem is a pain in the neck in leap.
Best of luck!
Sergio Aragon
Professor of Chemistry
San Francisco State University
-----Original Message-----
From: Sindrila Dutta banik [mailto:sindrila.duttabanik.yahoo.com]
Sent: Tuesday, June 14, 2011 10:57 PM
To: amber.ambermd.org
Subject: [AMBER] Problem related to the load PDB
Hello!
I have a crystal structure of an enzyme (HisRS) which is tetramer. The residues between 185 to 228 are missing for all chains. How to manage this issue?
If I load the pdb file as it is then it forms a bond between the residue 185 and 228 which is unnatural. A part of the pdb file showing the coordinate of residue 185 and residue 228 is shown as follows:
ATOM 1793 N LEU A 185 2.488 44.813 7.408 1.00 77.39 N
ATOM 1794 CA LEU A 185 3.537 43.802 7.492 1.00 80.42 C
ATOM 1795 C LEU A 185 4.779 44.212 6.665 1.00 82.35 C
ATOM 1796 O LEU A 185 5.855 44.529 7.215 1.00 83.14 O
ATOM 1797 CB LEU A 185 2.993 42.429 7.043 1.00 79.48 C
ATOM 1798 H LEU A 185 1.645 44.592 6.981 1.00 0.00 H
ATOM 1799 N TYR A 228 -12.077 46.733 12.305 1.00 60.84 N
ATOM 1800 CA TYR A 228 -12.163 46.419 10.872 1.00 61.23 C
ATOM 1801 C TYR A 228 -13.202 45.304 10.589 1.00 61.67 C
ATOM 1802 O TYR A 228 -13.126 44.609 9.561 1.00 61.76 O
ATOM 1803 CB TYR A 228 -10.757 46.026 10.324 1.00 60.27 C
ATOM 1804 H TYR A 228 -12.893 46.851 12.828 1.00 0.00 H
If I add ‘TER’ in between the coordinates of residue 185 and coordinate of residue 228 then leap consider the 185 residue as C-terminal residue and 228 residue as N-terminal residue and creates one additional positive charge for the amino croup of 185 residue and a negative charge for the carboxylic acid group of 228 residue. A part of the pdb file showing the coordinate of residue 185 and residue 228 in the modified pdb file is shown as follows:
ATOM 1793 N LEU A 185 2.488 44.813 7.408 1.00 77.39 N
ATOM 1794 CA LEU A 185 3.537 43.802 7.492 1.00 80.42 C
ATOM 1795 C LEU A 185 4.779 44.212 6.665 1.00 82.35 C
ATOM 1796 O LEU A 185 5.855 44.529 7.215 1.00 83.14 O
ATOM 1797 CB LEU A 185 2.993 42.429 7.043 1.00 79.48 C
ATOM 1798 H LEU A 185 1.645 44.592 6.981 1.00 0.00 H
TER
ATOM 1799 N TYR A 228 -12.077 46.733 12.305 1.00 60.84 N
ATOM 1800 CA TYR A 228 -12.163 46.419 10.872 1.00 61.23 C
ATOM 1801 C TYR A 228 -13.202 45.304 10.589 1.00 61.67 C
ATOM 1802 O TYR A 228 -13.126 44.609 9.561 1.00 61.76 O
ATOM 1803 CB TYR A 228 -10.757 46.026 10.324 1.00 60.27 C
Now I want to make the carboxylic acid group (-COO-) of 185 residue and amino group of (-NH3+) residue 228 as neutral. How to do this? I also want to know in general what is used in such cases.
With best regards
Sindrila
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Received on Wed Jun 15 2011 - 00:00:04 PDT
