Practical – Analysis of Protein Families

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 navigating into it.

mkdir

The mkdir command stands for “make directory” and 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 tp4
cd tp4

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.

Characterization of a Sequence of Unknown Function

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

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 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)

Exercise 2 How many hits did you get with E ≤ 10?

Exercise 3 To which family does the protein seem to belong?

Exercise 4 According to BLASTP alignments, propose 2 patterns conserved in the protein family.

Exercise 5 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 6 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?

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

Solution

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

The sequence of Poribacteria genome is available at: /data/poribacteria/poribacteria_genomes.fasta

Create the BLAST database with the makeblastdb command.

To see available options:

makeblastdb –help

Solution

makeblastdb -in /data/poribacteria/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

Solution

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

Analysis of the TblastN Results.

Display the TblastN output:

more selN.tblastn

Exercise 7 Can we extend the gene sequence in 3’?

Exercise 8 What do you learn from this search about the evolution of this gene in available Poribacteria genomes?

Exercise 9 We obtain several alignments for the sequence VYCT01000218. Why?

For that, use grepcommand with -A option. The -A option tells grep to display not only the matching line but also the subsequent n lines after each match.

Solution

grep -A 90 '>VYCT01000218' selN.tblastn

Analysis of a Protein Family

A protein sequence (available on the Moodle platform) has been automatically predicted during the annotation of the fungus Malassezia globosa genome.

Similarity Search

Perform a similarity search using BLAST at EBI in the SWISSPROT database (https://www.ebi.ac.uk/jdispatcher/sss/ncbiblast) (default parameters).

Exercise 10 How many sequences do you obtain (with E<10)? Why so few sequences are detected according to you?

Display results with Tool output.

Exercise 11 Find 3 conserved motifs.

Exercise 12 Is the Malassezia sequence homologous to: Q84424, P05469, P0C995, O13881?

Display results with Visual output.

Exercise 13 For each group of homologs, specify which region(s) (N-terminus, central region, C-terminus, almost whole sequence…) of the query sequence and of the database sequences have been aligned by BLAST.

Exercise 14 Why can this information be important before constructing a global multiple alignment?

Display the Summary table. Select all sequences detected by BLAST except:

CLR2_SCHPO (O13881) and YKP3_KLULA (P05469): non-homologous sequences
YL226_MIMIV (Q5UQC0) and MCE_MIMIV (Q5UQX1): too few motifs.

Save sequences in Fasta format and add the query sequence in the file. The final file is also available on Moodle (distant_homologs.txt).

Multiple Alignment of Homologs Using Mafft

Parameters:

Display alignment parameters.

Exercise 15 Why are pairwise parameters mentioned?

Align sequences (Alignment, Do complete alignment) with default parameters.

Visualize this alignment with Jalview (available on your computer or using https://www.jalview.org/jalview-js/JalviewJS/).

Residues can be colored according to their properties with Colour, Zappo (or Clustal, for instance).

Exercise 16 Is the sequence order relevant? Modify sequence order if necessary.

Exercise 17 Do all sequences exhibit the same domain organization?

Exercise 18 Do you find the previously defined motifs? According to you, is the alignment correct?

Exercise 19 Can you find additional motifs? Can you find obvious sequence prediction errors?

Exercise 20 Which sequence sub-groups can be defined? Are there “orphan” sequences? Justify your answer and give one discriminative residue or motif for each group.