Dear Henry,

This one is slightly tricky. Below is a tutorial no how to do it. The key 
point is that you need to start from the 'Sequences' tab when you add the 
molecule. If you start from 'Add Sequence' the program will automatically 
add a chain etc. and that makes it impossible to modify the molecule 
afterwards. Confusing, I know, but 'Add Sequence' was meant to be a single 
command to do all the operations at once. That is nice for the normal 
case, it just does not allow for un-normal things like adding disulphides.

Yours,

Rasmus

Tutorial follows:

NB It ws taken from the middle of a longer tutorial. It works (I tested 
it) but just ignore comments about other chains etc. that should have been 
there. The tutorial creates a single two-chain molecule. It should be 
obvious how to change it to make two chains from two different molecules.


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.

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:Molecules:Molecule Setup 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. 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 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. 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 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 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. 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).

---------------------------------------------------------------------------
Dr. Rasmus H. Fogh                  Email: [log in to unmask]
Dept. of Biochemistry, University of Cambridge,
80 Tennis Court Road, Cambridge CB2 1GA, UK.     FAX (01223)766002

On Sun, 24 Jan 2010, Henry Hocking wrote:

> Hello,
>
> I want to add disulfide links to my new template molecule but it seems that 
> in Release 2.1.2 newly created template molecules are locked by default.
> How do I unlock it and is there a way to change this default preference?
>
> Cheers,
>
> Henry