TITLE: Sequence alignment for Biochemistry I
AUTHORS: Belle Houston, Chris Berndsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Comparing DNA or protein sequences can provide insight into the structure or function of newly discovered or characterized open reading frames or proteins. The amino acid sequence is often </span><span style = "font-weight:bold;">conserved</span><span> between species or between proteins with similar structural/functional properties. A sequence that is conserved means that the sequence is identical across several species. A sequence can have aspects that are  similar meaning that while the amino acids are not identical, they have similar properties. Examples of similar sequences are an D --> E change or L --> I change. </span></div><div class = "text-block">The video below provides a brief overview of the process:</div><div style = "text-align :; float : ;"><video style = "" src = "https://s3.amazonaws.com/protocols-files/public/5917da60180611bbcc4c7255f94ad072ace49c2e3114d13b111e60a40a9364c1/b2y2bb58f.mp4" /></div></div>

[STEPS]
?. [Obtain Sequence from Uniprot]
Obtain sequence from Uniprot OR extract sequence from structure in YASARA (skip to step 3)
Some things to note about the sequence you use:If you obtain sequences from Uniprot, these are full sequences of proteins where the structure may not be entirely known for all amino acids. If you extract the sequence from YASARA, the sequence will most likely have less amino acids than the Uniprot sequence as it is only providing the seqeunce of amino acids in the known structure. No matter which method you use, be sure to know the species that the sequence originates from.For Video Summary see abstract
?. [BLAST your Protein ]
Use the NCBI BLAST page to BLAST your sequence.
BLAST = Basic Local Alignment Search Tool; it will search various sequence data bases to find matche to your sequences and those that are similar, which may be useful for finding proteins with similar structure or function.
?. [Shade the alignment using Boxshade]
Use BoxShade to better see the similarities and differences across the orthologs. Click here to open the BoxShade home page.
?. [Align the sequences with Clustal Omega]
Use Clustal Omega to align the sequences.  Click here for the ClustalOmega homepage
?. [Obtain Sequence from Uniprot]
For Uniprot click hereClick on the link above and locate your protein on Uniprot by using the search bar. In this procedure, we will be using the UBA5 protein found in humans as an example protein.
?. [Obtain Sequence from Uniprot]
Your search results will pull up proteins from multiple oranisms. Be sure to chose the protein from the correct organism. In this case, we are interested in the UBA5 in humans. If your protein is found in a plant such as Arabisopsis, be sure it says that in the organism column.
?. [Obtain Sequence from Uniprot]
You should be able to locate avaliable sequences of your protein by looking at the options on the left and clicking the 'sequences tab'. Or, scroll down until you find the seqeunces. The available sequences will be shown. Sometimes they will provide different isoforms of the protein. Be sure to research which isoform you are interested in if there are different isoforms. Press the 'FASTA' download button as shown above the seqeunce.
?. [Obtain Sequence from Uniprot]
Once you press the FASTA download button, a page like the one shown below should come up. Copy the ENTIRE text starting at the >. This text is to be pasted into the NCBI pBLAST decribed in the next steps.
?. [BLAST your Protein ]
Sometimes it is useful to narrow your search by restricting the database or excluding organisms. THIS IS OPTIONAL!The example below shows how to restrict the data base to model organisms and exclude human sequences, however this is not required for sequence alignment to work.Select the 'Model Organism (landmark)' in the dropdown menu under 'Choose Search Set'. Exclude Hominidae (taxid:9604) from the search. Be sure to check the exclude box!
?. [BLAST your Protein ]
Select 'pBlast' for the Program Selection.
blastp is the most general search but also least adventurous search tool. PSI- or PHI-BLAST can return more hits by changing the algorithm and the weighting of sequence variation. While useful for finding distantly related proteins, it can result in some very different sequences being included. Use these with caution and experience.
?. [BLAST your Protein ]
Press 'Blast'It may take a few minutes to a few hours before you see results.
?. [BLAST your Protein ]
Once the results have appeared, you can se the check boxes to the left to select sequences of your protein found in different species. Select options that have a query cover of 50% and above or so. Use proteins with less query coverage or less than 50% sequence identity with caution
Proteins that serve the same function but are in different species are called orthologs. Some of the options available are only predicted to exist indicated as PREDICTED or hypothetical protein. Avoid these as these are not yet confirmed to exist.
?. [BLAST your Protein ]
Once you have your desired sequences chosen, press 'Download' >> FASTA (Complete Sequence)
?. [BLAST your Protein ]
These sequences should appear in your downloads. They will open with Notepad for PC users. Copy all the text.
?. [Align the sequences with Clustal Omega]
Paste your BLAST result sequences into the 'sequences in any supported format:' box
?. [Align the sequences with Clustal Omega]
IMPORTANT! The formatting for the sequences is sensitive. At the end of every sequence, press the 'Enter' key to make a space between each sequence.
?. [Align the sequences with Clustal Omega]
Select 'ClustalW with character counts' for the output file.
?. [Align the sequences with Clustal Omega]
Click 'Submit'. It may take a minute for it to produce results. Your results should look like those below.
?. [Align the sequences with Clustal Omega]
Copy from the word 'Clustal' at the top of the alignment down the last character in the alignment.
?. [Shade the alignment using Boxshade]
Select 'RTF_new' for 'Output format' and 'ALN' for 'input sequence format'. Paste your ClustalOmega results into the box at the bottom.
?. [Shade the alignment using Boxshade]
Press 'Run BOXSHADE...' . It may take a couple of minutes.
?. [Shade the alignment using Boxshade]
When it is done, you will get an output window. Press on the 'Output number 1' link provided. This will download the BoxShade file. It can be opened with Microsoft Word.
?. [Shade the alignment using Boxshade]
Analyze your results in Word. Black highlight means perfect conservationGrey highlight mean an amino acid or a position with some conservationNo highlight means no conservationA dash means a gap or missing amino acid in that sequence
?. [BLAST your Protein ]
Paste the seqeunce you've copied from either UNIPROT or YASARA into the box under the 'Enter Query Sequence' tab.
?. [Do you have a sequence already?]
If you are getting the sequence from Uniprot, go to Step 2If you are extracting the sequence from a PDB file, go to Step 3If you have a sequence already provided skip to Step 4.
?. [Obtain Sequence from a PDB file]
Extracting sequence from object on YASARAOpen your structure of choice on YASARA. Once the object is loaded, click the 'Analyze' tab at the top of the page, then 'Sequence of', then 'Object'.
?. [Obtain Sequence from a PDB file]
When the following window pops up, be sure to select the correct object. The following objects have their PDB ID's listed. The 6H77 selected is the PDB ID for the model of UBA5 being analyzed in this example.
?. [Obtain Sequence from a PDB file]
The sequences should come up in the command bar at the bottom. Highlight your sequence. It will appear red once you highlight it. Press 'Cntrl C' (PC) or 'Command C' (Mac) to copy this sequence.