Entering non-standard molecules
Now we will look again at setting up molecular information in a CCPN project, but this time we we go beyond the canonical linear protein sequence and enter some of the non-standard connectivities and residues. This means we have to use a slightly different procedure, that allows us to modify the molecule template before we create the Mol System. The popups and most of the operations are the same, but because we are starting in a different way, the program behaviour is different.
We will setup a discontinuous molecule with two polypeptide sections and internal disulphide links. This would be the situation that you would find in insulin for example. To enter a molecule start by going to M: Molecule: Molecules and select the {Sequences} tab. At the top change the "Molecule:" pulldown menu to "<New>" then click [Add Polymer] and accept the name for the molecule by clicking [OK]. You are now taken to the {Add Sequence} tab. Ensure that the Input Type is set to "1-letter" and type in any arbitrary protein sequence, with the sole constraint that it must have two cysteine residues(e.g. whatcheck). Then click [Add Sequence!] and you will be taken back to the {Sequence} tab where you can see the section of polypeptide you have just created. Now click [Add Polymer] once again and for a second time add another protein sequence with two cysteine residues (e.g. wcheck) and press [Add Sequence!]. When you return once again to the {Sequence} tab you will see that there are two polypeptide regions, and by looking in the "Polymer Linking" and "Linked Residues" columns you can see that the sections are separate.
For example, for the entered sequences "whatcheck" and "check" this gives:
Now find the row of the first Cys residue and double-click in the "Descriptor & Stereochemistry" cell. Change the descriptor from "prot:HG" to "link:SG" (for non-terminal Cys residues). Repeat this for the three remaining Cys residues. This is done to say that all of the Cys residues are disulphide bonded, rather than carrying an SH group. Then with a Cys row selected click on [Edit Links]. You are now taken to the {Links} tab with the appropriate Cys selected. You will see that the "prev" and "next" links will be filled in with existing residues along th epolypeptide chain, but the "SG" link has no destination residue set. Double-click in the "Destination Residue" column for the "SG" row and set the residue to one of the Cys residues from the other polypeptide section. We have linked two Cys residues by a disulphide link but have still one more link to make. In the "Source Residue" pulldown select one of the unlinked Cys residues and set its destination residue to the last unlined Cys. Returning to the {Sequence} column you will see that the Cys residues are listed as having three linked residues; two from the peptide and one from disulphide.
We will now use our fully linked molecule, which is really just a sequence template, to build a chain containing all of the atoms that can be used for NMR assignment. Accordingly select the {Chains} tab and ensure that the "Mol System for new chain:" pulldown is set to "<New>" and that the "Template for new chain" is set to the disulphide linked molecule we just created. Using a new molecular system is important here so that we keep the new sequence separate from the existing protein. Now click [Make Chain From Template] and accept the MolSystem code and chain code by pressing [OK]. You will see that a new chain has appeared in the top table, but unlike the existing protein chain it has two chain fragments (for "whatcheck" and "wcheck"):
Click on the row of the new chain and you will see that the bottom table changes to show the two polypeptide regions. Now we will change the numbering of the second polypeptide section of our chain. Do this by double-clicking the |Start Seq Number| column for the second row. Now enter a number that is higher than the original start number.
Finally we will look at the fruits of our labour by selecting M: Molecule: Atom Browser. In the Chain pulldown menu select the last entry, which should correspond to our newly entered sequence, and ensure that the hydrogen atoms are visible by clicking the [H] button. Firstly have a look at the Cys residues, you will see that they have no gamma hydrogen, which is what we would expect given the disulphide links. Then scroll down in the table to look at the end of the first polypeptide region and the beginning of the second. Note that the Residue number is discontinuous after we set a different starting number for the second section (here, the start of the second region is set to 13):
Note that we could have also changed the starting number of the first section too, as long as there is no overlap with the second (i.e. residue numbers must be unique).