​Using the internet, find the program Clustal Omega

PART C: Making the sequence alignment:
1. ​Using the internet, find the program Clustal Omega ​http://www.ebi.ac.uk/Tools/msa/clustalo/
a.​ Copy and paste your word/text file into the box with ALL 10 sequences, ​1PTH needs
to be at the top.
b.​ Select the ​more options ​button under STEP 2 – Set your parameters. Change the
drop-down menu under ORDER to read ​Input.
c.​ Click on ​Submit
d.​ Select ​Download Alignment File
e. ​Print this ​file (should open in a new window)
(9) State the purpose of Clustal Omega. State what the dashed lines represent in the clustalw
(aln) output.
2. ​You will now analyze a section of the 1PTH sequence to find residues (​residue​ is word for an
amino acid in a protein sequence) that are potentially structurally and/or functionally important
by looking for conservation! ​The section that we will focus on is V95 – L200
(10) State the importance of conserved amino acids in a sequence alignment. What information
do researchers mine from amino acid sequence alignments?
a.​ The following will help you find columns of 80 – 100% conserved amino acids in the
appropriate color. Do this by hand on the printed copy of your alignment!
b. ​Make a color key using four colors of your choice for the different amino acid types.
Here is an example:
Pink = polar, positively charged amino acids (a.a.)
Green = polar, negatively charged a.a.
Orange = polar, uncharged a.a
Blue = nonpolar, hydrophobic a.a
c.​ Include your color key to the side of the sequence alignment
d. ​Go through each column of the sequence and highlight with the appropriate color
ONLY those columns that are 80 – 100 % conserved with the 1PTH residue. Again only
need focus on this portion of the sequence alignment: ​V95 – L200
i.​ Example: If the column has 8 out of 10 (or 9 or 10 out of 10) amino acids that
are the same type (ie all polar positive) you would highlight the column pink.
Note in this does NOT mean the entire column will be the exact same amino acid.
Authors: Drs. Laura Listenberger and Cassidy Terrell Last updated: 12 February 2019
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In this instance, you might highlight a column where 8, 9 or 10 out of 10 resides
are “K” and “R.”
ii. ​You are trying to find the residues in 1PTH’s sequence that are conserved, so
when analyzing for conservation you MUST include and compare to the 1PTH
residue.
iii.​ A dash counts as a “miss.” Example: If the column has three dashes and the
rest of the amino acids are all “K” or “R” – this column has 70% conservation and
would not be highlighted.
(11) Pick 5 residues in 1PTH that are likely to be important for the structure and/or function.
Circle the residues in 1PTH on your sequence alignment. For each residue, predict if the residue
is likely to be on the surface or in the interior core of the protein.
(12) Explain why it is important to have a variety of species for a sequence alignment. Consider
what a sequence alignment from closely related species would look like.
(13)** Turn in a ​scanned​ PDF copy of your completed sequence alignment to the course website
or where your instructor indicates. Include your color key and the percent conservation assumed
while highlighting.
When finished, you can leave the Clustal Omega website