Entering non-standard molecules

Introduction

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. 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.

Creating a molecule with linked disulphide bridges

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 a protein sequence with two cysteine residues (e.g. CHECK) 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.

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". Repeat this for the three remaining Cys residues. For "WHATCHECK+CHECK" this would look like:

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, 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 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 many times listed as having three linked residues; two from the peptide and one from disulphide. For "WHATCHECK+CHECK" with links between residue 5 and 10 and between residue 8 and 13 this would look like:

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, go to {Chains} 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 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], then answer [Yes] if Analysis asks about equivalent aromatic atoms. You will see that a new chain has appeared in the top table, but unlike the existing protein chain it has two chain fragments.

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: Molecules: 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. For the example with WHATCHECK+CHECK with the starting residue number set to 11 for the second section, this would look like:

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).

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