ARIA disulphides

How to incorporate disulphide bonds in ARIA driven calculations based on a CCPN project.

ARIA 2.3 and CCPNMR analysis 2.1.5

ARIA 2.3 ignores disulphide bonds in CCPNMR projects and so these need to be incorporated in the ARIA project itself. There are two ways to do this: either incorporate them as specified covalent bonds; or as distance restraints that can optionally be ambiguous.

Generally the ambiguous route is more likely to be successful as it reduces the topological constraint on the structures at early stages of the calculation resulting in fewer failed structures, less knotting, etc. so that is the method described here.

Generate a table of restraints

Generate CNS distance restraints to hold the appropriate half-cystine SG atoms together. These restraints will be treated as NOE-like distance restraints in the calculations and use the sum version of the NOE potential (with exponent 20?) by default. You can therefore write ambiguous restraints if you are unsure of your disulphide bonding pattern, but want to enforce disulphides in any case. It is probably safer to leave the disulphides unrestrained until you can pair them up though!

An unambiguous restraint would be of the following form

 assi

   (( segid "   A" and resid 64   and name  SG  ))

   (( segid "   A" and resid 120  and name  SG  ))

      2.020     0.100     0.100

for the specified residues in segid (chain) A. Use a .tbl file extension when you save the file.

Load restraints into CCPN project

Load your distance restraints via formatConverter M:Import:Single files:Distance constraints:CNS then select your constraints file. You will be asked to select a Structure generation to add the restraints to - this corresponds to a Restraint set in analysis 2.1.5 terminology. Once you have done this and named the restraint list, formatConverter will suggest you run linkResonances - click [Yes] to this and the question about using the default settings, and in the Link chains dialogue select |Link to code '' (all)| unless you have more than one chain.

Add a Disulphide bridges (restraints) entry

From the ARIA gui main menu Add..:Disulphide Bridges will create a new entry under Disulphide bridges (restraints) where you can select the restraint list from the CCPN project (Format: CCPN, CCPN id: Select..).

You should also enter the residue numbers and segids in the Cysteines involved table. This has the effect of patching the description of the structure to remove the HGs modify the SGs and add bond angle and dihedral angle restraints from the forcefield.

Ensure ARIA CNS protocols are typo-free

In an earlier ARIA 2.3.1 release the refine_water.inp protocol contains a typographical error that results in the restraints not being included in the final water refinement. Check that line 91 reads "ambiguous".