The Calculate Mutation Energy (Binding) and Calculate Mutation Energy (Stability) are two very useful protocols for calculating the difference between the folding free energy of the wild type and mutated structure in Discovery Studio. The user specifies a set of selected residues in their protein structure and a list of residues types each selected residue should be mutated to plus whether Single, Double, Triple or All Selected Residues mutation sites should be explored.

One needs to be a bit careful when setting up these jobs since the number of mutants generated is combinatorial and can easily explode into an unreasonable number of calculations.

• In the case of the Single Mutations, it is straightforward. For each selected residue you add up the number of types chosen. For example, select 3 residues and for each residue you choose 4 types you will generate 12 mutants.
• In the case of the Double Mutations and Triple Mutations, the following formula is used:
  

[N!/r!(N-r)!]( M^r)

N = the number of selected residues to mutate

M = the number of amino acid types to mutate to

r = 2 for double mutations and 3 for triple mutations

Let’s say you have a protein that you want to look at 6 residues at the active site and perform double mutations for the 7 representative amino acid types. In this case r is 2, N is 6 and M is 7 and the resulting number of mutants generated is 735. 

If you select 15 residues and perform triple mutations for all 20 amino acid types (r is 3, N is 15 and M is 20) then the number of mutants is 3,640,000!!

• In the case of the All Selected Residues, the formula is N^M. So, if there are 4 residues selected and each is mutated to 3 amino acid types the total number of mutants would be 81. 

Hopefully, that helps you decide which parameter settings to choose before you hit Run.