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Auxiliary workflow to run SWAAT annotation on non-ADME genes

The auxiliary workflow will help in building the database required to annotate other non ADME genes that are not available within the default settings that we have provided with SWAAT.

Dependencies

All the dependencies are open source. The user needs only to obtain a free licence for FoldX. We recommend installing the dependencies using conda virtual environment. We also recommend using the Mamba package manager which will speed up the building process.

A special consideration must be paid to the installation of ENCoM. ENCoM can be obtained from its git repository. The build_encom code systematically returns an exit status of 1 which creates an issue when running within a Nextflow process. To overcome the problem, you need to modify line 370 from return(1); to return(0); in src/build_encom.c. Thereafter you can compile the set of codes as following:

cd ENCoM/build/ \
	&& make -f Makefile.Unix

Input data

The user is required to provide:

  • A list of Uniprot IDs corresponding to the genes to annotate
  • Molecular structures in PDB file format corresponding to the proteins encoded by the genes to annotate.

Providing the list of genes

The Uniprot list must be arranged in a text file containing no header as one accession per line. No header or hanging empty lines should be provided in the file. A typical layout is the following.

O75795
P20813
P33261
Q16348

Preparing the PDB structures

Follow these guidance to prepare the structures.

  • The structure must have a single chain.
  • The PDB file must be named as follows <UNIPROTID>.pdb (e.g. P04637.pdb )
  • A structure must contain a chain ID.
  • Heteroatoms are not taken into account
  • No discontinuities are allowed in the protein chain.

The MMTSB tool set is could be very handy to prepare the protein structure.

Calculation of the PSSMs

The process that runs this task uses the standalone version of PRODRES. The user must set the corresponding options in the command line or in the workflow file. However, if PRODRES is not installed, the generation of the PSSMs will not take place, However, Nextflow won't complai about this. Notice also that the PSSMs are mandatory when using the main workflow of annotation. We have made this choice because the installation of PRODRES may be non trivial for the user. SWAAT and the auxiliary workflow use the protein sequences of the Refseq database.

However, the user can download the FASTA Refseq sequences of the proteins, modify their header lines and and submit them to the server version of PRODRES. However, we recommand running the auxiliary workflow first which will prepare the FASTA sequences for you, and then submit them to the server as a multi sequence FASTA file/string.

FTMap input data

FTMap is a lool (doi.org/10.1038/nprot.2015.043 ) used in the Identification of binding hot spots. This is the only step that will require a manual processing by the user. However, if the gene is not known to bind drugs or small ligands, it won't be necessary to go through this step. The user needs to submit the structure to the STMap server and then download the 'hbonded' and the 'nonbonded' raw output output files to a clean location with the gene symbol as the name for the directory, The files should be tagged "nonbonded" and "hbonded" strings (e.g. mage6_hbonded.rawextract and mage6_nonbonded.rawextract) The location of the parent directory, i.e. containing the gene symbol named folders, can then be specified in the ftmap option.

Running the workflow

Options of the workflow can be listed using the following command.

nextflow run prepare_db.nf --help

A typical running of the workflow is as follows:

nextflow run prepare_db.nf --protlist /path/to/Uniprot_list.txt \
	--pdbs /path/to/pdb/files
	--outfolder /path/to/the/outputfolder

Output

The auxiliary workflows generates a series of directories, each containing data that are used by the main workflow to speed-up the annotation process.

  • sequences: Contains the FASTA sequences extracted from Refseq and Uniprot.
  • uniprot2PDBmap contains the mapping of the reference sequence protein coordinates to the PDB sequence coordinates.
  • Seq2Chain links the gene symbol to Uniprot ID, the chain identifier and the PDB file name.
  • prot_annotation offers information about the functional contribution of amino acids in the reference sequence.
  • maps contains the mapping between the gene coordinates and the protein reference sequence.
  • hotspots contains the identified hotspot information relative to the PDB coordinates.
  • ENCom contains the normal modes calculated for the reference protein structure.
  • PSSMs contains the pssm files calculated for the protein using the reference sequence.
  • PDBs contains the 3D structures in PDB file format.
  • ftmap contains scored amino acid positions for ligand/drug binding putative site.

Example: Melanoma-associated antigen 6 (MAGE-6)

The Uniprot accession for MAGE-6 is P43360. The structure of this protein contains one chain. The structure were generated using homology modeling and provided in the directory input_example/PDB as P43360.pdb.

Next you need a text file containing no header in the following format

P43360

The file can also be found in the input_example directory (example.csv) .

The FTMap files were calculated by submitting the PDB structure to the servers which results in two output files. They were renamed to contain the tags "hbonded" and "nonbonded". The raw files were put into a directory MAGEA6 named after the gene symbol (this is a requirement). The MAGEA6 directory is a child directory of input_example/ftmap. We can then use the option --ftmap to point to the location of the ftmap raw files.

One you have prepared the inputs you can call the auxiliary workflow as follows:

nextflow run prepare_db.nf --protlist /home/hothman/SWAAT/auxiliary_wf/input_example/example.csv \
	--pdbs /home/hothman/SWAAT/auxiliary_wf/input_example/PDB \
	--outfolder /home/hothman/new_genes_database
	--ftmap /home/houcem/SWAAT/auxiliary_wf/input_example/ftmap

A successful run will create a new folder containing the database directory (new_genes_database) that are necessary to annotate the MAGE-6 gene using structural data. The output of the command should give something similar to the following:

Number of genes    : 1
Path to PDB files  : /home/houcem/SWAAT/auxiliary_wf/input_example/PDB
Outputdir          : database_new_gene/
List of accessions : P43360
executor >  local (8)
[d9/9641a8] process > GetProteinAnnotationFetchFasta (1) [100%] 1 of 1 ✔
[61/310184] process > GetCoordinates (1)                 [100%] 1 of 1 ✔
executor >  local (9)
[d9/9641a8] process > GetProteinAnnotationFetchFasta (1) [100%] 1 of 1 ✔
[61/310184] process > GetCoordinates (1)                 [100%] 1 of 1 ✔
[42/fb35d6] process > geneToChainMapping (1)             [100%] 1 of 1 ✔
[58/0178c7] process > uniprot2PDB (1)                    [100%] 1 of 1 ✔
[ab/c18a85] process > Hospotislands (1)                  [100%] 1 of 1 ✔
[6a/76b297] process > encomWT (1)                        [100%] 1 of 1 ✔
[04/7ba5b9] process > parseFTMAP (1)                     [100%] 1 of 1 ✔
[a3/4dca9d] process > generate_matrixes                  [100%] 1 of 1 ✔
[d8/c557ae] process > outputPDB (1)                      [100%] 1 of 1 ✔
Completed at: 06-Nov-2021 14:15:35
Duration    : 1m 53s
CPU hours   : (a few seconds)
Succeeded   : 9