Difference between revisions of "Task6 Hemochromatosis Protocol"

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== Amino acid features ==
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Casting Spell: Create Protocol Page
  
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The values were taken from [http://en.wikipedia.org/wiki/Amino_acid Wiki: Amino Acids] and [http://en.wikipedia.org/wiki/Proteinogenic_amino_acid Wiki: Proteinogenic Amino Acids].
  +
  +
<br style="clear:both;">
  +
  +
== BLOSUM62/PAM1/PAM250 ==
  +
  +
  +
* BLOSUM62: [http://www.ncbi.nlm.nih.gov/Class/BLAST/BLOSUM62.txt NCBI: BLAST] (last accessed 17th Jun 2012)
  +
  +
A R N D C Q E G H I L K M F P S T W Y V B Z X *
  +
A 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0 -2 -1 0 -4
  +
R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3 -1 0 -1 -4
  +
N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3 3 0 -1 -4
  +
D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 4 1 -1 -4
  +
C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 -3 -3 -2 -4
  +
Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2 0 3 -1 -4
  +
E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2 1 4 -1 -4
  +
G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 -1 -2 -1 -4
  +
H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3 0 0 -1 -4
  +
I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3 -3 -3 -1 -4
  +
L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1 -4 -3 -1 -4
  +
K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2 0 1 -1 -4
  +
M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1 -3 -1 -1 -4
  +
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1 -3 -3 -1 -4
  +
P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2 -2 -1 -2 -4
  +
S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2 0 0 0 -4
  +
T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0 -1 -1 0 -4
  +
W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3 -4 -3 -2 -4
  +
Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1 -3 -2 -1 -4
  +
V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 -3 -2 -1 -4
  +
B -2 -1 3 4 -3 0 1 -1 0 -3 -4 0 -3 -3 -2 0 -1 -4 -3 -3 4 1 -1 -4
  +
Z -1 0 0 1 -3 3 4 -2 0 -3 -3 1 -1 -3 -1 0 -1 -3 -2 -2 1 4 -1 -4
  +
X 0 -1 -1 -1 -2 -1 -1 -1 -1 -1 -1 -1 -1 -1 -2 0 0 -2 -1 -1 -1 -1 -1 -4
  +
* -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 1
  +
  +
  +
* PAM1: [http://www.icp.ucl.ac.be/~opperd/private/pam1.html http://www.icp.ucl.ac.be] (last accessed 17th Jun 2012)
  +
  +
Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
  +
A R N D C Q E G H I L K M F P S T W Y V
  +
Ala A 9867 2 9 10 3 8 17 21 2 6 4 2 6 2 22 35 32 0 2 18
  +
Arg R 1 9913 1 0 1 10 0 0 10 3 1 19 4 1 4 6 1 8 0 1
  +
Asn N 4 1 9822 36 0 4 6 6 21 3 1 13 0 1 2 20 9 1 4 1
  +
Asp D 6 0 42 9859 0 6 53 6 4 1 0 3 0 0 1 5 3 0 0 1
  +
Cys C 1 1 0 0 9973 0 0 0 1 1 0 0 0 0 1 5 1 0 3 2
  +
Gln Q 3 9 4 5 0 9876 27 1 23 1 3 6 4 0 6 2 2 0 0 1
  +
Glu E 10 0 7 56 0 35 9865 4 2 3 1 4 1 0 3 4 2 0 1 2
  +
Gly G 21 1 12 11 1 3 7 9935 1 0 1 2 1 1 3 21 3 0 0 5
  +
His H 1 8 18 3 1 20 1 0 9912 0 1 1 0 2 3 1 1 1 4 1
  +
Ile I 2 2 3 1 2 1 2 0 0 9872 9 2 12 7 0 1 7 0 1 33
  +
Leu L 3 1 3 0 0 6 1 1 4 22 9947 2 45 13 3 1 3 4 2 15
  +
Lys K 2 37 25 6 0 12 7 2 2 4 1 9926 20 0 3 8 11 0 1 1
  +
Met M 1 1 0 0 0 2 0 0 0 5 8 4 9874 1 0 1 2 0 0 4
  +
Phe F 1 1 1 0 0 0 0 1 2 8 6 0 4 9946 0 2 1 3 28 0
  +
Pro P 13 5 2 1 1 8 3 2 5 1 2 2 1 1 9926 12 4 0 0 2
  +
Ser S 28 11 34 7 11 4 6 16 2 2 1 7 4 3 17 9840 38 5 2 2
  +
Thr T 22 2 13 4 1 3 2 2 1 11 2 8 6 1 5 32 9871 0 2 9
  +
Trp W 0 2 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 9976 1 0
  +
Tyr Y 1 0 3 0 3 0 1 0 4 1 1 0 0 21 0 1 1 2 9945 1
  +
Val V 13 2 1 1 3 2 2 3 3 57 11 1 17 1 3 2 10 0 2 9901
  +
  +
  +
* PAM250: [http://www.icp.ucl.ac.be/~opperd/private/pam250.html http://www.icp.ucl.ac.be] (last accessed 17th Jun 2012)
  +
  +
Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
  +
A R N D C Q E G H I L K M F P S T W Y V
  +
Ala A 13 6 9 9 5 8 9 12 6 8 6 7 7 4 11 11 11 2 4 9
  +
Arg R 3 17 4 3 2 5 3 2 6 3 2 9 4 1 4 4 3 7 2 2
  +
Asn N 4 4 6 7 2 5 6 4 6 3 2 5 3 2 4 5 4 2 3 3
  +
Asp D 5 4 8 11 1 7 10 5 6 3 2 5 3 1 4 5 5 1 2 3
  +
Cys C 2 1 1 1 52 1 1 2 2 2 1 1 1 1 2 3 2 1 4 2
  +
Gln Q 3 5 5 6 1 10 7 3 7 2 3 5 3 1 4 3 3 1 2 3
  +
Glu E 5 4 7 11 1 9 12 5 6 3 2 5 3 1 4 5 5 1 2 3
  +
Gly G 12 5 10 10 4 7 9 27 5 5 4 6 5 3 8 11 9 2 3 7
  +
His H 2 5 5 4 2 7 4 2 15 2 2 3 2 2 3 3 2 2 3 2
  +
Ile I 3 2 2 2 2 2 2 2 2 10 6 2 6 5 2 3 4 1 3 9
  +
Leu L 6 4 4 3 2 6 4 3 5 15 34 4 20 13 5 4 6 6 7 13
  +
Lys K 6 18 10 8 2 10 8 5 8 5 4 24 9 2 6 8 8 4 3 5
  +
Met M 1 1 1 1 0 1 1 1 1 2 3 2 6 2 1 1 1 1 1 2
  +
Phe F 2 1 2 1 1 1 1 1 3 5 6 1 4 32 1 2 2 4 20 3
  +
Pro P 7 5 5 4 3 5 4 5 5 3 3 4 3 2 20 6 5 1 2 4
  +
Ser S 9 6 8 7 7 6 7 9 6 5 4 7 5 3 9 10 9 4 4 6
  +
Thr T 8 5 6 6 4 5 5 6 4 6 4 6 5 3 6 8 11 2 3 6
  +
Trp W 0 2 0 0 0 0 0 0 1 0 1 0 0 1 0 1 0 55 1 0
  +
Tyr Y 1 1 2 1 3 1 1 1 3 2 2 1 2 15 1 2 2 3 31 2
  +
Val V 7 4 4 4 4 4 4 4 5 4 15 10 4 10 5 5 5 72 4 17
  +
  +
<br style="clear:both;">
  +
  +
== Secondary structure ==
  +
  +
  +
Sequence and PyMol editing was performed by hand. The SwissModel were created with the [http://swissmodel.expasy.org/workspace/index.php?func=modelling_simple1 WebServer] (template: 1a6zC).
  +
  +
<br style="clear:both;">
  +
  +
== PSSM-Matrix and homolog search ==
  +
  +
The PSSM-Matrix and homolog search was done by using PSI-Blast with the command:
  +
blastpgp -i Q30201.fasta -j 5 -Q outMatrix.txt -o output.txt -d /mnt/project/pracstrucfunc12/data/big/big
  +
  +
To retrieve all homologous proteins, we retained only the results of the fifth iteration and used the following script to extract them:
  +
  +
  +
<source lang="perl">
  +
#!usr/bin/perl
  +
  +
use strict;
  +
#use warnings;
  +
#use autodie;
  +
my $file= shift;
  +
  +
open (FILE, "<$file");
  +
while (<FILE>){
  +
my $currentLine=$_;
  +
chomp $currentLine;
  +
  +
if ($currentLine=~m/^..\|(.+)\|.*\d\d\d.*/gi){
  +
print "$1\n";
  +
}
  +
}
  +
</source>
  +
  +
Because we only wanted mammalian protein sequences, we used Unipot to retrieve the corresponding information to these protein by getting the Flat-formatted file.
  +
  +
To get only mammalian proteins we used the following script on the flat file:
  +
  +
  +
  +
<source lang="perl">
  +
#!usr/bin/perl
  +
  +
use strict;
  +
#use warnings;
  +
#use autodie;
  +
my $file= shift;
  +
  +
my @currentIDs="";
  +
open (FILE, "<$file");
  +
while (<FILE>){
  +
my $currentLine=$_;
  +
chomp $currentLine;
  +
  +
if ($currentLine=~m/^AC\s(.*)/gi){
  +
$currentLine=$1;
  +
# print "$currentLine 1\n";
  +
$currentLine=~s/\s//gi;
  +
  +
  +
push(@currentIDs, split(/;/,$currentLine));
  +
#print "$1\n";
  +
} elsif ($currentLine=~m/^SQ\s(.*)/gi){
  +
@currentIDs="";
  +
} elsif ($currentLine=~m/^OC.*Mammalia/gi){
  +
# print "Mammalian found, bitches!\n";
  +
foreach my $entry (@currentIDs){
  +
if ($entry ne ""){
  +
print "$entry\n";
  +
}
  +
}
  +
}
  +
}
  +
</source>
  +
  +
This way we got all mammalian protein IDs that are homologs to our protein and retrieved their sequences through Uniprot.
  +
  +
As some proteins have multiple IDs noted in the flat file we retained our PSIBlast found IDs by using the
  +
uniq -d
  +
command.
  +
  +
After adding our own sequence, we used these sequences in ClustalW and Muscle to retrieve the MSAs.
  +
  +
clustalw -align -infile=Mammals.fasta -outfile=MammalsClustalw.fasta
  +
muscle -in Mammals.fasta -out MuscleMSAMammals.fasta

Latest revision as of 17:17, 18 June 2012

Amino acid features

The values were taken from Wiki: Amino Acids and Wiki: Proteinogenic Amino Acids.


BLOSUM62/PAM1/PAM250

   A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V  B  Z  X  *
A  4 -1 -2 -2  0 -1 -1  0 -2 -1 -1 -1 -1 -2 -1  1  0 -3 -2  0 -2 -1  0 -4 
R -1  5  0 -2 -3  1  0 -2  0 -3 -2  2 -1 -3 -2 -1 -1 -3 -2 -3 -1  0 -1 -4 
N -2  0  6  1 -3  0  0  0  1 -3 -3  0 -2 -3 -2  1  0 -4 -2 -3  3  0 -1 -4 
D -2 -2  1  6 -3  0  2 -1 -1 -3 -4 -1 -3 -3 -1  0 -1 -4 -3 -3  4  1 -1 -4 
C  0 -3 -3 -3  9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 -3 -3 -2 -4 
Q -1  1  0  0 -3  5  2 -2  0 -3 -2  1  0 -3 -1  0 -1 -2 -1 -2  0  3 -1 -4 
E -1  0  0  2 -4  2  5 -2  0 -3 -3  1 -2 -3 -1  0 -1 -3 -2 -2  1  4 -1 -4 
G  0 -2  0 -1 -3 -2 -2  6 -2 -4 -4 -2 -3 -3 -2  0 -2 -2 -3 -3 -1 -2 -1 -4 
H -2  0  1 -1 -3  0  0 -2  8 -3 -3 -1 -2 -1 -2 -1 -2 -2  2 -3  0  0 -1 -4 
I -1 -3 -3 -3 -1 -3 -3 -4 -3  4  2 -3  1  0 -3 -2 -1 -3 -1  3 -3 -3 -1 -4 
L -1 -2 -3 -4 -1 -2 -3 -4 -3  2  4 -2  2  0 -3 -2 -1 -2 -1  1 -4 -3 -1 -4 
K -1  2  0 -1 -3  1  1 -2 -1 -3 -2  5 -1 -3 -1  0 -1 -3 -2 -2  0  1 -1 -4 
M -1 -1 -2 -3 -1  0 -2 -3 -2  1  2 -1  5  0 -2 -1 -1 -1 -1  1 -3 -1 -1 -4 
F -2 -3 -3 -3 -2 -3 -3 -3 -1  0  0 -3  0  6 -4 -2 -2  1  3 -1 -3 -3 -1 -4 
P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4  7 -1 -1 -4 -3 -2 -2 -1 -2 -4 
S  1 -1  1  0 -1  0  0  0 -1 -2 -2  0 -1 -2 -1  4  1 -3 -2 -2  0  0  0 -4 
T  0 -1  0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1  1  5 -2 -2  0 -1 -1  0 -4 
W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1  1 -4 -3 -2 11  2 -3 -4 -3 -2 -4 
Y -2 -2 -2 -3 -2 -1 -2 -3  2 -1 -1 -2 -1  3 -3 -2 -2  2  7 -1 -3 -2 -1 -4 
V  0 -3 -3 -3 -1 -2 -2 -3 -3  3  1 -2  1 -1 -2 -2  0 -3 -1  4 -3 -2 -1 -4 
B -2 -1  3  4 -3  0  1 -1  0 -3 -4  0 -3 -3 -2  0 -1 -4 -3 -3  4  1 -1 -4 
Z -1  0  0  1 -3  3  4 -2  0 -3 -3  1 -1 -3 -1  0 -1 -3 -2 -2  1  4 -1 -4 
X  0 -1 -1 -1 -2 -1 -1 -1 -1 -1 -1 -1 -1 -1 -2  0  0 -2 -1 -1 -1 -1 -1 -4 
* -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4  1 



        Ala  Arg  Asn  Asp  Cys  Gln  Glu  Gly  His  Ile  Leu  Lys  Met  Phe  Pro  Ser  Thr  Trp  Tyr  Val
          A    R    N    D    C    Q    E    G    H    I    L    K    M    F    P    S    T    W    Y    V
Ala A  9867    2    9   10    3    8   17   21    2    6    4    2    6    2   22   35   32    0    2   18
Arg R     1 9913    1    0    1   10    0    0   10    3    1   19    4    1    4    6    1    8    0    1
Asn N     4    1 9822   36    0    4    6    6   21    3    1   13    0    1    2   20    9    1    4    1
Asp D     6    0   42 9859    0    6   53    6    4    1    0    3    0    0    1    5    3    0    0    1
Cys C     1    1    0    0 9973    0    0    0    1    1    0    0    0    0    1    5    1    0    3    2
Gln Q     3    9    4    5    0 9876   27    1   23    1    3    6    4    0    6    2    2    0    0    1
Glu E    10    0    7   56    0   35 9865    4    2    3    1    4    1    0    3    4    2    0    1    2
Gly G    21    1   12   11    1    3    7 9935    1    0    1    2    1    1    3   21    3    0    0    5
His H     1    8   18    3    1   20    1    0 9912    0    1    1    0    2    3    1    1    1    4    1
Ile I     2    2    3    1    2    1    2    0    0 9872    9    2   12    7    0    1    7    0    1   33
Leu L     3    1    3    0    0    6    1    1    4   22 9947    2   45   13    3    1    3    4    2   15
Lys K     2   37   25    6    0   12    7    2    2    4    1 9926   20    0    3    8   11    0    1    1
Met M     1    1    0    0    0    2    0    0    0    5    8    4 9874    1    0    1    2    0    0    4
Phe F     1    1    1    0    0    0    0    1    2    8    6    0    4 9946    0    2    1    3   28    0
Pro P    13    5    2    1    1    8    3    2    5    1    2    2    1    1 9926   12    4    0    0    2
Ser S    28   11   34    7   11    4    6   16    2    2    1    7    4    3   17 9840   38    5    2    2
Thr T    22    2   13    4    1    3    2    2    1   11    2    8    6    1    5   32 9871    0    2    9
Trp W     0    2    0    0    0    0    0    0    0    0    0    0    0    1    0    1    0 9976    1    0
Tyr Y     1    0    3    0    3    0    1    0    4    1    1    0    0   21    0    1    1    2 9945    1
Val V    13    2    1    1    3    2    2    3    3   57   11    1   17    1    3    2   10    0    2 9901



        Ala  Arg  Asn  Asp  Cys  Gln  Glu  Gly  His  Ile  Leu  Lys  Met  Phe  Pro  Ser  Thr  Trp  Tyr  Val
          A    R    N    D    C    Q    E    G    H    I    L    K    M    F    P    S    T    W    Y    V
Ala A    13    6    9    9    5    8    9   12    6    8    6    7    7    4   11   11   11    2    4    9
Arg R     3   17    4    3    2    5    3    2    6    3    2    9    4    1    4    4    3    7    2    2
Asn N     4    4    6    7    2    5    6    4    6    3    2    5    3    2    4    5    4    2    3    3
Asp D     5    4    8   11    1    7   10    5    6    3    2    5    3    1    4    5    5    1    2    3
Cys C     2    1    1    1   52    1    1    2    2    2    1    1    1    1    2    3    2    1    4    2
Gln Q     3    5    5    6    1   10    7    3    7    2    3    5    3    1    4    3    3    1    2    3
Glu E     5    4    7   11    1    9   12    5    6    3    2    5    3    1    4    5    5    1    2    3
Gly G    12    5   10   10    4    7    9   27    5    5    4    6    5    3    8   11    9    2    3    7
His H     2    5    5    4    2    7    4    2   15    2    2    3    2    2    3    3    2    2    3    2
Ile I     3    2    2    2    2    2    2    2    2   10    6    2    6    5    2    3    4    1    3    9
Leu L     6    4    4    3    2    6    4    3    5   15   34    4   20   13    5    4    6    6    7   13
Lys K     6   18   10    8    2   10    8    5    8    5    4   24    9    2    6    8    8    4    3    5
Met M     1    1    1    1    0    1    1    1    1    2    3    2    6    2    1    1    1    1    1    2
Phe F     2    1    2    1    1    1    1    1    3    5    6    1    4   32    1    2    2    4   20    3
Pro P     7    5    5    4    3    5    4    5    5    3    3    4    3    2   20    6    5    1    2    4
Ser S     9    6    8    7    7    6    7    9    6    5    4    7    5    3    9   10    9    4    4    6
Thr T     8    5    6    6    4    5    5    6    4    6    4    6    5    3    6    8   11    2    3    6
Trp W     0    2    0    0    0    0    0    0    1    0    1    0    0    1    0    1    0   55    1    0
Tyr Y     1    1    2    1    3    1    1    1    3    2    2    1    2   15    1    2    2    3   31    2
Val V     7    4    4    4    4    4    4    4    5    4   15   10    4   10    5    5    5   72    4   17


Secondary structure

Sequence and PyMol editing was performed by hand. The SwissModel were created with the WebServer (template: 1a6zC).


PSSM-Matrix and homolog search

The PSSM-Matrix and homolog search was done by using PSI-Blast with the command: 

blastpgp -i Q30201.fasta -j 5 -Q outMatrix.txt -o output.txt -d /mnt/project/pracstrucfunc12/data/big/big

To retrieve all homologous proteins, we retained only the results of the fifth iteration and used the following script to extract them:


<source lang="perl">

  1. !usr/bin/perl

use strict;

  1. use warnings;
  2. use autodie;

my $file= shift;

open (FILE, "<$file"); while (<FILE>){ my $currentLine=$_; chomp $currentLine;

if ($currentLine=~m/^..\|(.+)\|.*\d\d\d.*/gi){ print "$1\n"; } } </source>

Because we only wanted mammalian protein sequences, we used Unipot to retrieve the corresponding information to these protein by getting the Flat-formatted file.

To get only mammalian proteins we used the following script on the flat file:


<source lang="perl">

  1. !usr/bin/perl

use strict;

  1. use warnings;
  2. use autodie;

my $file= shift;

my @currentIDs=""; open (FILE, "<$file"); while (<FILE>){ my $currentLine=$_; chomp $currentLine;

if ($currentLine=~m/^AC\s(.*)/gi){ $currentLine=$1;

  1. print "$currentLine 1\n";

$currentLine=~s/\s//gi;


push(@currentIDs, split(/;/,$currentLine)); #print "$1\n"; } elsif ($currentLine=~m/^SQ\s(.*)/gi){ @currentIDs=""; } elsif ($currentLine=~m/^OC.*Mammalia/gi){ # print "Mammalian found, bitches!\n"; foreach my $entry (@currentIDs){ if ($entry ne ""){ print "$entry\n"; } } } } </source>

This way we got all mammalian protein IDs that are homologs to our protein and retrieved their sequences through Uniprot.

As some proteins have multiple IDs noted in the flat file we retained our PSIBlast found IDs by using the 

uniq -d

command.

After adding our own sequence, we used these sequences in ClustalW and Muscle to retrieve the MSAs. 

clustalw -align -infile=Mammals.fasta -outfile=MammalsClustalw.fasta
muscle -in Mammals.fasta -out MuscleMSAMammals.fasta
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