Practical – Analysis of Protein Families

1. Characterization of a Sequence of Unknown Function

> Hypothetical protein [Candidatus Poribacteria sp. WGA-A3]
MVDFNNTLSSTASVQVAAHWDPIENTANNLHRSTEEKFNRDKAQWQEPVEMTWEQWLEVFNPGPAHPLKN
YSTADFQVFLPPSTVNVADVWDLDTEGILPFLRQFHPSATMKLPRYGSIPSDQKDAKACLRALSPEYADI
VFRIHARFTLDASIDAYFMPAQFAGHLIINRNSRTIHQWTLSLPNRNSNVDIGAFRSHDIGFVPRMELCS
VSETQPESIVWEAAITAEEADKKFQNSLYKFAEIEWTPIEEAVELAKASNRSIHAVLLFGVLDDESC

First Part: BLASTP Search

Search for the organism Candidatus Poribacteria in the NCBI Taxonomy database.

Exercise 1 What can you say about the organism taxonomy?

Search for homologs of the Poribacteria sequence (available on Moodle) using NCBI BLASTP:

In the RefSeq_protein database (not in RefSeq select!!!)
With word size = 3
With a maximum of 1000 sequences and E ≤ 10 (other parameters: default)
How many hits did you get with E ≤ 10?

Exercise 2 To which family does the protein seem to belong?

Exercise 3 According to BLASTP alignments, propose 2 patterns conserved in the protein family.

Exercise 4 In which taxonomic group(s) do we find hits? Are the hits found in the bacterial sequences significant? Propose a function and an evolutionary origin for the gene encoding the Poribacteria protein.

Exercise 5 Look at the alignment with human selenoprotein N NP_996809.1. Do the alignments cover the C-terminal part of the query sequence and animal selenoprotein N? In metazoans, which amino acid “matches” the stop codon of the Poribacteria sequence? What hypothesis can be made? How could this hypothesis be tested?

Connection to the server

To learn how to connect to the server for practical work, refer to the Server Connection Guide.

Once you’re connected, start by creating a working directory for this session and navigate into it.

mkdir

The mkdir command stands for “make directory”, is used to create a new folder in the file system. You can create your directory like this:

mkdir <directory_name>

The cd command stands for “change directory”, allows you to move into a different folder within the file system. Use it as follows:

cd <directory>

Solution

mkdir TdBlast
cd TdBlast

Help

If you need help with a command, display its help using: - <command-h or <command--help for short help messages. - man <command> to display the full manual of a command.

Comparison Protein/Genome Using Command-Line BLAST

Retrieve the Human Protein Sequence of the Selenoprotein N (Accession Number: Q9NZV5) using the command GET of the UniProtKB API.

Documentation of UniProtKB API: UniProtKB API Documentation

curl -X GET --header 'Accept:text/x-fasta' -o Q9NZV5.fasta 'https://www.ebi.ac.uk/proteins/api/proteins?offset=0&size=100&accession=Q9NZV5'

You can open the file with the cat command:

cat Q9NZV5.fasta

Create a BLAST Database with Poribacteria Genomes

Copy the fasta file containing sequences from 2 Poribacteria genomes to your directory. The sequence is available at: /home/lecompte/Poribacteria_genomes.fasta

cp /home/lecompte/Poribacteria_genomes.fasta Poribacteria_genomes.fasta

Create the BLAST database with the makeblastdb command: To see available options:

makeblastdb –help

makeblastdb -in Poribacteria_genomes.fasta -dbtype nucl -out PoribacteriaDb

Perform a Similarity Search using the human selenoprotein N Q9NZV5.fasta as a query in the PoribacteriaDb database using the tblastN command: To see available options:

tblastn –help

tblastn -db PoribacteriaDb -query Q9NZV5.fasta -out selN.tblastn

Analysis of the TblastN Results.

Display the TblastN output:

more selN.tblastn

Exercise 6 Can we extend the gene sequence in 3’?

Exercise 7 What do you learn from this search about the evolution of this gene in available Poribacteria genomes?

Exercise 8 We obtain several alignments for the sequence VYCT01000218. Why?

grep -A 90 '>VYCT01000218' selN.tblastn