Preparing Your Own Input
========================

Preparing Your Input MSA and PDB with Colabfold
-----------------------------------------------

You have a fasta file for your protein of interest and you want to understand
impact of (certain) mutations.
Before starting, please make sure that your fasta file does not contain a gap.
The quickest method to obtain both multiple sequence alignment and a protein
structure is to use Colabfold. Let's do this step by step:

1. Let's go the Colabfold web site:

   https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb

   Sign in using your gmail account.

2. Click on the 'Connect' button on the top right hand side.

3. Clean 'query_sequence' box and paste your sequence to the 'query_sequence' box.
   For me, I selected adenylate kinase (AKE) as my example fasta sequence
   (https://www.rcsb.org/fasta/entry/4AKE/display).

4. Change the 'jobname' to something that makes more sense to you.

5. Go to the menu bar of your 'AlphaFold2.ipynb' notebook, where 'File, Edit,
   View, Insert, Runtime, Tools, Help' are listed. Click on the Runtime and
   select 'Run all'. 

6. This process make take from a few minutes to a few hours depending on your
   protein size. It will give you an a3m file and up to 5 PDB models. Put these
   files in a clean folder and change the directory to that folder in your
   terminal. 
   
7. Unfortunately, a3m file is not in fasta format and it contains gap columns. 
   We have to clean those gaps. We can do that with a GUI program like Ugene
   or Jalview. However, it is a labor intensive procedure. Here, I will use a
   small tool that I developed and added to the PRESCOTT docker image that I created.
   
8. Start the docker image with the following command:

   .. code:: bash

	     sudo docker run -ti --rm --mount type=bind,source=$PWD,target=/home/tekpinar/research/myexample \
	     tekpinar/esgemme-docker:v1.3.0

9. Now, change the directory to myexample folder. 

   .. code:: bash

	     cd ../myexample/
	     ls -l

   We are supposed to see our a3m and pdb files in this folder.

10. Let's use a small script from hhsuite to convert a3m file to fasta format.

    .. code:: bash
	      
	      reformat.pl a3m fas AKE.a3m AKE.fasta

11. Final step and we are there:

    .. code:: bash

	      demust removegaps -i AKE.fasta -o AKE_nogaps.fasta

There is one last step to reach our goal. ID and description parts of the
a3m and fasta files are too long. We have to shorten them. We can do that with

 .. code:: bash

   awk 'BEGIN{FS=" "}{if(NF>1) {printf(">%s\n", $1)}else{print $0}}' AKE_nogaps.fasta > AKE_nogaps_short_names.fasta
   # Recheck this command if you can remove extra >


Congratulations! Now, you have all the input files required for PRESCOTT:
I. An input MSA: AKE_nogaps_short_names.fasta
II. An input PDB: myprotein.pdb



	 
