TITLE: Homology modeling using Phyre2 for Biochemistry I
AUTHORS: Michael Friedman, Chris Berndsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for homology modeling proteins for use in Biochemistry I at James Madison University. Protocol guides students to use the PHYRE2 web server (citation below). </div><br/><br/><div class = "text-block">Citations for servers:</div><div class = "text-block"><ol style = "list-style-type: decimal;"></ol></div></div>

[STEPS]
?. [NCBI BLAST]
Navigate to NCBI BLAST (Basic Local Sequence Alignment Tool) and paste your sequence into the "Enter Query Sequence" box.
?. [NCBI BLAST]
The standard settings for the search are shown in the table.  ABC1Default SettingWhat it does2Enter Query Sequence3Query Subrange(Blank)Limits search to a part of the sequence. Can be useful if there are common motifs/domains in the sequence.4Choose Search Set5DatabaseNon-redundant protein sequences (nr)Limits search to a sub-set of sequences. For homology modeling searching the Protein Data Bank proteins (pdb) is a good idea if you want to see if your modeling might be successful.6Organism(Blank)Limit search to a specific organism or other taxonomic group.7Exclude(Unchecked)Reduce results by removing certain classifications of sequences.8Program Selection9AlgorithmblastpSetting changes how the databases are searched. blastp is the most straight-forward. PSI-BLAST is useful when the query sequence is not easily aligned to other sequences.1011
ABC1Default SettingWhat it does2Enter Query Sequence3Query Subrange(Blank)Limits search to a part of the sequence. Can be useful if there are common motifs/domains in the sequence.4Choose Search Set5DatabaseNon-redundant protein sequences (nr)Limits search to a sub-set of sequences. For homology modeling searching the Protein Data Bank proteins (pdb) is a good idea if you want to see if your modeling might be successful.6Organism(Blank)Limit search to a specific organism or other taxonomic group.7Exclude(Unchecked)Reduce results by removing certain classifications of sequences.8Program Selection9AlgorithmblastpSetting changes how the databases are searched. blastp is the most straight-forward. PSI-BLAST is useful when the query sequence is not easily aligned to other sequences.1011
?. [NCBI BLAST]
Record any changes to the settings in Step 2.1 below:
?. [NCBI BLAST]
Press BLAST and wait until the results return.Thie search can take up to
[hour]
?. [Analysis of BLAST results to ID sequence]
Results will be returned as shown as below:
?. [Analysis of BLAST results to ID sequence]
Column definitions from the Descriptions tab of the results.  AB1Table columnWhat it tells you2DescriptionTells you identify of matching sequence. Predicted or hypothetical in title indicates protein has not been verified.3Max ScoreDuring alignment identities, similarities, and gaps are scored. This indicates the best score if the sequence was aligned multiple times.4Total ScoreIf many disconnected parts matched, this is the sum of the max scores for those5Query CoverIndicates the percentate of the query sequence found in the match. 100% means all of the sequence was found.6E valueE(xpect) value tells you how many sequences that would rank higher if this was a random match. 0 or very small numbers are good.7Per. IdentHow much of the sequence was identical in sequence. Need >40% for good homology model.8AccessionThe accession number for the sequence. Can be clicked to take you to the info card on that sequence.
AB1Table columnWhat it tells you2DescriptionTells you identify of matching sequence. Predicted or hypothetical in title indicates protein has not been verified.3Max ScoreDuring alignment identities, similarities, and gaps are scored. This indicates the best score if the sequence was aligned multiple times.4Total ScoreIf many disconnected parts matched, this is the sum of the max scores for those5Query CoverIndicates the percentate of the query sequence found in the match. 100% means all of the sequence was found.6E valueE(xpect) value tells you how many sequences that would rank higher if this was a random match. 0 or very small numbers are good.7Per. IdentHow much of the sequence was identical in sequence. Need >40% for good homology model.8AccessionThe accession number for the sequence. Can be clicked to take you to the info card on that sequence.
?. [Analysis of BLAST results to ID sequence]
Record your best 5 sequences and their statistics in the table below.  ABCDEFG1Sequence DescriptionMax ScoreTotal ScoreQuery CoverageE valuePer IdentAccession23456
ABCDEFG1Sequence DescriptionMax ScoreTotal ScoreQuery CoverageE valuePer IdentAccession23456
?. [Analysis of BLAST results to ID sequence]
In the Graphic Summary tab, you can view the domains in your sequence. A domain is a part of the sequence with a known fold/shape/structure. A motif is a sequence that has a shape or function. Typically domains can fold on their on, while motifs are shorter pieces within domains.
?. [Analysis of BLAST results to ID sequence]
Record any domains or motifs in the table below along with the approximate position within the sequence. This can help in the modeling and support the accuracy of your model later on.  AB1Domain/Motif nameposition (this should be a number/set of numbers)23456
AB1Domain/Motif nameposition (this should be a number/set of numbers)23456
?. [Analysis of BLAST results to ID sequence]
In the Alignments tab, the actual sequence alignment (the data) are shown.
?. [Analysis of BLAST results to ID sequence]
Each alignment shows the following key information:Identities and their location within the sequence.Positives and their location within the sequence.Gaps and their location within the sequence.The alignment: Your sequence is the top row, the matched sequence in the middle row (+ means similar), and the sequece from the database (called Sbjct). Position number of the sequence match. These are the numbers at each end of the sequences.
?. [Analysis of BLAST results to ID sequence]
Press the Download link to the top right of the alignment and select Text you will get a complete file of your results. Upload this to your project folder for this project and name the file:[date]_[sequence_name]_[team_name]_BLAST_alignment.txtReplace [team_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.
?. [Analysis of BLAST results to ID sequence]
Indicate your file location as a link within a note on this step.  THIS IS YOUR DATA FILE FOR THE SEARCH!
?. [Analysis of BLAST results to ID potential modeling templates]
and repeat search but limit the Database to Protein Data Bank proteins (pdb). This search will identify proteins of known structure that match your protein and can suggest if your modeling attempt will be successful. Record your sequence matches in the table.
?. [Analysis of BLAST results to ID potential modeling templates]
Accession numbers here lead to the information on the structure which may help when using SWISS-MODEL. These accession numbers are the PDB ID numbers.  ABCDEFG1Sequence DescriptionMax ScoreTotal ScoreQuery CoverageE valuePer IdentAccession23456
ABCDEFG1Sequence DescriptionMax ScoreTotal ScoreQuery CoverageE valuePer IdentAccession23456
?. [Analysis of BLAST results to ID potential modeling templates]
The top five structures here are potential templates structures which you can use to model your sequence. This means these structures are similar at the sequence level to your sequence and potentially  will result in a similar structure to your sequence.
?. [Homology Modeling]
Having identified the sequence and potential templates, now it is possible to start modeling the sequence to generate a potential sequence.
?. [Homology Modeling]
Go to the Phyre2 server. This should take you to a page that looks like this.
?. [Homology Modeling]
Paste your sequence into the Amino Acid Sequence box as shown above.
?. [Homology Modeling]
Provide your:email address so the results and model can be sent to youA job description so you can keep track of your dataWhich mode you want to use. Intensive takes longer but can give better results for models with few templates. Choose normal unless you identified less than 3 templates from BLAST.Select NOT for profit if you are a JMU studentRecord your job description in this step as a note.
?. [Homology Modeling]
Something like this will appear. Your results will be sent to you via email. Time to retrieve the result varies depending on the server but usually is more than
?. [Analysis of Phyre2 results]
Here is a sample of the results linked in the emailed results. Make sure to download this model for comparison. If you find that there are other models that this was built from that you prefer feel free to use their links for comparison too!
?. [Analysis of Phyre2 results]
Note the percentage of residues modelled and the location of low confidence regions from the scheme in the Confidence Summary box.  AB1Percent of residues modeled:2Low confidence region locations:3
AB1Percent of residues modeled:2Low confidence region locations:3
?. [Analysis of Phyre2 results]
Download the model and the zip of all results and upload these files into your project folder. Name the .pdb file as:[date]_[sequence_name]_[team_name]_PHYRE_model.pdbReplace [team_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.  [date] with the date in YYYYMMDD formatName the .zip file as:PHYRE_results_[Group_name]_[sequence_name].zipReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.Ex. 07162021_CRX-DBD_Berndsen_PHYRE_model.pdb
?. [Analysis of Phyre2 results]
Indicate your file location as a link within a note on this step.  THIS IS YOUR DATA FILE FOR THE PHYRE modeling!
?. [Analysis of Phyre2 results]
In the Sequence analysis section, you can download the sequence alignment file used in the modeling.
?. [Analysis of Phyre2 results]
The Secondary structure and disorder prediction section, you can see what the predicted secondary structure is along with the confidence in that prediction (9 is high, 0 is low). Also, the disorder prediction is shown with ? suggesting disorder and the confidence in that prediction (9 is high, 0 is low). A PDF of this figure can be download using the symbol on the left.
?. [Analysis of Phyre2 results]
Upload your PDF to your project folder.Name the .pdf file as:[date]_[sequence_name]__[Group_name]_PHYRE_SecStrPred.pdfReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.
?. [Analysis of Phyre2 results]
Indicate your file location as a link within a note on this step.
?. [Analysis of Phyre2 results]
In the Domain Analysis section, you can move the cursor over each red part of the aligned region and see the predicted domains.
This should match the domains identified in step 3!
?. [Analysis of Phyre2 results]
Record the code and the domain name for the top 5 hits in the table.  AB1CodeDomain/motif23456
AB1CodeDomain/motif23456
?. [Analysis of Phyre2 results]
In the Detailed Template information table, there is important information about the templates.
?. [Analysis of Phyre2 results]
Take a screen shot of the table showing the top 5 hits and upload the photo to your project folder/notebook.Name the file as:[date]_[sequence_name]_[Group_name]_PHYRE_templateinfoReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.
?. [Analysis of Phyre2 results]
Indicate your file location as a link within a note on this step.
?. [Analysis of Phyre2 results]
Save your record, export it as a PDF, and place it in the your notebook files. If this is part of the modeling project, make sure that you also modeled using SWISS-model