ddG of multiple mutations on a monomer

Hello all!

I've been attempting to see the effect of mutating several surface residues on a small protein to arginines, and have been trying to use Rosetta to assess how well the fold will tolerate these changes. I have a crystal structure of the protein, and my idea was to use the ddG Monomer application to do so. These arginine mutations will eventually all be implemented into the protein, and so I would ideally like to know each individual ddG of these mutations, but also the ddG of having all of the mutations present. I know the information I may obtain from this isn't at all a "real" manifestation of ddG, but so long as the fold doesn't completely die I think that would be more than enough to go forward with the project. Eventually we will do mutations accross the protein

I was able to get ddH Monomer running smoothly, preminimization and all, for both each individual single mutation and all the mutations at the same time.

However, I know Alford et. al used the cartesian ddG application for this in their score function paper. I also do know that the recent Frenz et. al. paper further explores this. However, I'm not sure I can get cartesian ddG running for multiple mutations? I was able to implement the single point mutants and obtain ddGs, but using a mutfile with all four mutations doesn't seem to have the desired effect. Using the following mutfile gives output below:

total 4
1
E 14 R
1
K 17 R
1
K 21 R
1
K 22 R

But using the following gives an error:

total 4
4
E 14 R
K 17 R
K 21 R
K 22 R

basic.io.database: (0) {0} Database file opened: scoring/score_functions/bondlength_bondangle/default-improper.txt
core.scoring.CartesianBondedEnergy: (0) {0} Read 2202 bb-independent improper tors.
protocols.CartesianddG: (0) {0}  interface ddg is 0
protocols.CartesianddG: (0) {0}  total read as 4

ERROR: Assertion `num_mutations>0` failed.
ERROR:: Exit from: src/protocols/ddg/CartesianddG.cc line: 293

[ ERROR ]: Caught exception:


File: src/protocols/ddg/CartesianddG.cc:293
[ ERROR ] UtilityExitException
ERROR: Assertion `num_mutations>0` failed.

I'm fairly certain K->R mutations won't be decidely untolerable, but would still like an estimation. Is this sort of calculation just not amenable for cartesian ddG? Using a single mutation or several different single point ones works and outputs correctly (see below mutfile.ddg.txt). Is this proper usage of this application or should I be approaching this differently?

Thanks!

-Héctor

EDIT: I think my confusion stems from the following paragraph in the cartesian_ddg application: " For ddg:mut_file format, please refer to here. Note that this file contains the mutations you want to introduce at once, which means, specifying more than one mutation in a single file will try to mutate all together at same time. Scanning over separate mutations (e.g. ALA scanning) will therefore require running this app separately using different mut_file as input."
 

Particularly since it doesn't seem that the mutations are being introduced at once, and they are not being mutated all together at the same time.