A multiple sequence alignment (MSA) file in fasta format (mandatory). Your query protein must be the first sequence in the fasta file. In addition, the query sequence should not contain any gaps.
A structure in PDB format (optional but highly recommended) of the query sequence.
The list of specific mutations should be a text with a mutation (e.g. A235P) per line.
One of the fastest ways to obtain both input MSA and a PDB file is to run colabfold: https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb
Please note that the MSA file produced by colabfold (a3m file) can contain gaps in the query sequence. You have to remove the corresponding columns from the alignment before using it in PRESCOTT. You can remove the gaps with programs that have a GUI, such as ugene (http://ugene.net/) or jalview (https://www.jalview.org/)..