(Answer based on further information – including the constraint file – provided during a private email exchange)
The issue is with how you’re specifying your secondary constraint for constraint block 2 and the difference between upstream and downstream. “Downstream” is the ligand that you’re trying to place. “Upstream” is the protein residue you’re making the constraint to. So when you say “SECONDARY_MATCH: UPSTREAM_CST 1” what you’re saying is that you want to do a secondary match against the protein residue (the upstream residue) from constraint block 1. But your constraint is written against your ICS ligand (the downstream residue). That’s the cause of the error – Rosetta is trying to match against the CYD residue from the first constraint block, but is using geometry specified from the ICS ligand residue. So Rosetta complains about not finding a suitable target_restype.
What you need to do is to use “SECONDARY_MATCH: DOWNSTREAM” instead. There’s only ever a single downstream residue, so you don’t need to specify the constraint number.
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