TITLE: Homology modeling 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 SWISS-Model and PHYRE2 web servers (citations below). </div><div class = "text-block"><span>The protocol directs users to save data in OSF or the </span><a href="http://osf.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Open Science Framework</span></a><span>. This is the preferred project management tool for the class and is required for JMU students using this for the course. Other users can use whichever system is preferred.  </span></div><div class = "text-block">Citations for servers:</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>Bertoni, M., Kiefer, F., Biasini, M., Bordoli, L., and Schwede, T. (2017) </span><span style = "font-style:italic;">Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology.</span><span> Sci. Rep. 7, 10480.  </span></li><li style = "counter-reset:ol0;"><span>Benkert, P., Biasini, M., and Schwede, T. (2011) </span><span style = "font-style:italic;">Toward the estimation of the absolute quality of individual protein structure models.</span><span> Bioinformatics 27, 343–350.  </span></li><li style = "counter-reset:ol0;"><span>Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., de Beer, T. A. P., Rempfer, C., Bordoli, L., Lepore, R., and Schwede, T. (2018) </span><span style = "font-style:italic;">SWISS-MODEL: homology modelling of protein structures and complexes. </span><span>Nucleic Acids Res. 46, W296–W303.</span></li><li style = "counter-reset:ol0;"><span>Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J. E. (2015) </span><span style = "font-style:italic;">The Phyre2 web portal for protein modeling, prediction and analysis. </span><span>Nat. Protoc. 10, 845–858.</span></li></ol></div></div>

[STEPS]
?. [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]
Follow the steps for the preferred server.
For the biochemistry course modeling project, both servers should be used.
?. [Homology Modeling]
Go to the Phyre2 server. This should take you to a page that looks like this.
?. [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
?. [homology modeling using SWISS-Model]
Click on the link for the SWISS-model server to get to a page that looks like
?. [Analysis of Phyre2 results]
Heres a sample of the results linked in the emailed results. Make sure to download this model for compairson. If you find that there are other models that this was built from that you prefer feel free to use their links for compairson too!
?. [homology modeling using SWISS-Model]
Make sure you have recorded all the required data. If you have completed the Phyre modeling. Save the record, export to PDF and upload this file to OSF in the Notebook files.
?. [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 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.
?. [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]
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 OSF folder for this project and name the file:BLAST_alignment_[Group_name]_[sequence_name].txtReplace [Group_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]
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]
Indicate your OSF file location as a link within a note on this step.  THIS IS YOUR DATA FILE FOR THE SEARCH!
?. [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.
?. [Analysis of Phyre2 results]
Download the model and the zip of all results and upload these files into OSF. Name the .pdb file as:PHYRE_model_[Group_name]_[sequence_name].pdbReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.Name 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.
?. [Analysis of Phyre2 results]
Indicate your OSF 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 Detailed Template information table, there is important information about the templates.
?. [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]
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 OSF.Name the .pdf file as:PHYRE_SecStrPred_[Group_name]_[sequence_name].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 OSF 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]
Take a screen shot of the table showing the top 5 hits and upload the photo to OSF.Name the file as:PHYRE_templateinfo_[Group_name]_[sequence_name]Replace [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 OSF 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 OSF folder for your notebook files. If this is part of the modeling project, make sure that you also modeled using SWISS-model
?. [homology modeling using SWISS-Model]
Follow the instructions on the image above to start the modeling by Build Model. Initial steps can take up to
Pressing Build Model is auto building using SWISS-Model with the best template according to SWISS-Model. Alternatively, you can Search for Templates and do template selection manually; guided by your templates identified using BLAST.
?. [homology modeling using SWISS-Model]
Once model building (either manual or automated) is complete, a screen as shown below will appear.
?. [homology modeling using SWISS-Model]
If you choose to do manual building via the Search for Templates tab. Record your templates below.  A1Template name (second column in table)2345
A1Template name (second column in table)2345
?. [homology modeling using SWISS-Model]
Useful information from this screenGMQE for Global Model Quality Estimation is scored from zero to 1 and indicates model quality based on the alignment with numbers closer to 1 indicating a more reliable model. QMEAN indicates the model quality based on structural features and the quality of the chemistry such as torsion angles and solvation. A good model has a number that is more positive, although a good model can have a negative QMEAN score. Less than -4 and model has bad chemistry.Local Quality Estimate indicates model quality on a per residue basis and can indicate if there are sections of hte model that are problematic (such as the ends of the model in the report above)Model-Template alignment shows how well the template structure and the sequence align and what parts of the model were used. Blue colors means better alignment while red colors mean worse alignment and modeling. Secondary structure is also indicated with tubes for α-helix and arrows for β-sheet.
?. [homology modeling using SWISS-Model]
The grey Model button leads to a menu to download information. Two key options:PDB format results in just the homology model, which can be viewed in YASARA or ChimeraModel Report downloads a .zip with the PDB file model and an HTML based report of the model process including the statistics shown in Step 8.1. Download both and upload both files to OSF. Name the PDB file:SWISS_model_[Group_name]_[sequence_name].pdbReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.Name the zip file:SWISS_data_[Group_name]_[sequence_name].zipReplace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.THESE ARE YOUR DATA FILES FOR SWISS MODEL!
?. [homology modeling using SWISS-Model]
Indicate your OSF file location as a link within a note on this step.
?. [homology modeling using SWISS-Model]
The Structure Assessment button leads to a new page showing a basic geometric and chemical assessment of the model.
?. [homology modeling using SWISS-Model]
The Ramachandran plot indicates if the phi/psi angles are appropriate for a protein structure and is interactive. The Phi angle is the dihedral angle for the rotation of the N-Cα bond while the psi angle is for rotation around the Cα-C bond of the amino acid backbone. The ideal angles for helices, sheets, and coils shown in green areas below are known to minimize steric clashes between atoms. Use the camera tool to record the Ramachandran plot and upload it to your OSF.Name the image file:SWISS_phipsi_[Group_name]_[sequence_name]Replace [Group_name] with your name/group name without the brackets. Replace [sequence_name] with the name of the sequence.  Add a note with the link to your file.
?. [homology modeling using SWISS-Model]
The Molprobity Results are numerical scores based on the model and indicate what percentage of amino acids that fall in the ideal geometry category and have minimal clashes. The check boxes allow for visualization of the bad amino acids and can be useful to see if there are general model problems or localized issues. Localized issues can be fixed, general problems cannot.
?. [homology modeling using SWISS-Model]
Record your Molprobity numbers.  ABC1Deviant amino acids2Molprobity Score3Clash Score4Ramachandran favored5Ramachandran outliers6Rotamer outliers7C-beta deviations8Bad bonds9Bad angles
ABC1Deviant amino acids2Molprobity Score3Clash Score4Ramachandran favored5Ramachandran outliers6Rotamer outliers7C-beta deviations8Bad bonds9Bad angles