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Prepare runs the Relax protocol, which aggressively minimizes backbone and side chain, while placing very tight constraints on every heavy (non-hydrogen) atom to keep the structure very close to the original crystal structure. So this is more restrained that a normal Relax run. Prepare will find a more favorable conformation in terms of Rosetta Energy. Often, this is a more natural conformation than many input structures because structures from the PDB are prone to unnatural variations due to crystal packing or experimental factors that cause low resolution in portions of the protein.

For Rosetta command-line users, this protocol uses