I have some questions about using enzyme design application for my system.
1. I have not used RosettaMatch, but I have a rough complex structure of the enzyme with the ligand. Is it fine to use that or I need to use RosettaMatch?
2. To generate the cst file for enzyme design applicaton, I have to use secondary match algorithms since I do not have the angle and dihedral information. I read Match documentation, but it is not clear to me is whrn to use UPSTREAM and when to DOWNSTREAM and also about the number is written in front of UPSTREAM.I know UPSTREAM is related to protein residues and DOWNSTREAM to ligand. Please clarify it for me with one example or refer me to a documentation about it that has an example?
3. In my system, other than the enzyme (chain A) and ligand (chain X), there is another protein (chain D) bound to the ligand. In other words, my substrate is composed of chain D and chain X, but only chain X is located in the binding pocket. I am thinking to define the covalent bond between chain D and chain X as one of the constraints in the cst file even it is not a catalytic residue. Is it correct to do that?
4. In this system, how can I make sure the binding energy is exactly between my enzyme (chain A) and my ligand (chain X)? I do not want chain D to be considered in binding energy. In enzyme design application documents, it is mentioned "If there are multiple protein chains, these figures may not accurately represent the total figures for binding."