For further information on this and other topics, please feel free to contact us at: academy@schrodinger.com

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MODELING

Building Homology Models with Prime: A Case Study with Factor Xa

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Building Homology Models with Prime: A Case Study with Factor Xa

In this tutorial we will use Prime to build homology models of the human protein factor Xa using two different templates, bovine factor Xa and human thrombin. The tutorial is divided into four sections:

1. Creating a project in Maestro2. Building models using the Structure Prediction Wizard 3. Assessing model quality 4. Refining models

Required File: factorXa_human.fasta, FXa.psipass2, FXa_homologymodelingexample.prjzip

MODELING

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1. Creating project files in Maestro

Before running any modeling tasks in Maestro, it is strongly recommended that you save your session as project (.prj file format). Once a project is created then any work you do will be automatically saved. An instance of the project can be exported as an archive (.prjzip or .prj.zip file format) for easy sharing with colleagues via email. Note: When archived projects are opened in Maestro, a new project must be created in order for any subsequent work on the project to be automatically saved.

1. Open an archived project. Start Maestro. In the Project toolbar, press the Open icon. From the working directory, select the file FXa_homologymodelingexample.prjzip; press Open. A scratch project will be created.

2. Create a new project. Save the scratch project by pressing the Save As icon in the Project toolbar. In the dialog box that appears, assign “FXa_homologymodeling” as the file name and press save. This will create a permanent project file; the header at the top of the Maestro window will change to FXa_homologymodeling.prj accordingly.

2. Building models using the Structure Prediction Wizard

Here we will build two different homology models of human Factor Xa using two different templates. The first model will be built in a straight-forward manner from a template structure of the bovine homolog of factor Xa (PDB 1KIG). The second model will be built from a human paralog, thrombin (PDB 2BVR). Since the sequence identify between human Factor Xa and thrombin is very low, we will use secondary structure information to guide manual alignment of the two sequences prior to building the final model. Model building consists of the following steps: (1) importing the target sequence, (2) identifying a model template(s), (3) editing the sequence alignment (if necessary), and (4) building the model. Additional refinement steps can be performed after the initial model is built (See Section 4).

3. Create a new run in the structure prediction wizard (SPW). Open the homology modeling window (via Tasks -> Homology Modeling in the Maestro menu bar); from the two choices that appear, select the Structure Prediction Wizard option (Figure 1). In the window that opens, create a new run (File -> New, Figure 1); you can use the default run name that appears and press Ok. The name of the run will be at the top of the window and the sequence areas will be blank.

Figure 1. The Homology Modeling (left side) and Structure Prediction Wizard (right side) windows.

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4. Import the target structure. Load the human FXa sequence by clicking the File button from Get sequence from; in the dialog box that opens, select FactorXa_human.fasta. Press Ok. The amino acid sequence will now appear in the upper window, while the name of the sequence will appear in the lower window (Figure 2).

Figure 2. View of the sequence windows after importing the FXa sequence. 5. Identify a model template. Press the Next button located at the bottom right hand corner of the window to progress to the Find Homologs step; here we will use BLAST to find a suitable template. Press the BLAST Homology Search button to initiate the search; when the job has finished, results will be displayed in the lower window, sorted in descending order based on their score . Displayed in the upper window will be the sequence alignment, colored by residue type, between FXa and the homolog that is highlighted in the lower window (Figure 3).

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Figure 3. Output of the homology search. Results are displayed in the lower window while the sequence alignment between FXa and the selected homolog is displayed in the top window.

In the lower results window, press the ID header to sort by ID; scroll down to 1KIG, a structure of a bovine FXa ortholog, and select it. Press Next to progress to the next step. 6. Edit the sequence alignment. In the upper sequence window, press the crop (scissors) icon in the toolbar, then highlight the region of target sequence that is not aligned to the template; this region of sequence should now be colored light grey and will be excluded from the model building step (Figure 4). Since the sequence identity is pretty high, we will not edit the alignment manually. Press Next to advance to the final model building step.

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Figure 4. The edit alignment step. Target sequence colored light grey will not be modeled.

7. Build the model. Keep the default options and press the Build Model button. Once the model is built, it will appear as a ribbon in the workspace (Figure 5). Residues that are identical to the template are colored purple, while residues that differ are colored cyan. Sequence insertions, if applicable, will be colored red. The model is automatically added to your project table.

Figure 5. Resulting model of human FXa built from 1KIG. Identical residues to the template are

colored purple while different residues are colored cyan.

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8. Generate an alternate model using 2bvr as the template

8A. Re-import the human FXa sequence and find homologs. Repeat steps 3 and 4 (i.e. create a new run and input the human FXa sequence). Advance to the Find Homologs step of the SPW and run a BLAST homology search; when the results appear, sort by ID and select 2BVR, a structure of the human paralog thrombin. Press Next to advance to the Edit Alignment step.

8B. Incorporate secondary structure information into the edit alignment step. Since the sequence identify between FXa and thrombin is pretty low, we are going to use secondary structure information to ensure optimal alignment of the two sequences. First, switch the Alignment Method to Prime STA. Since this approach utilizes secondary structure information to build the alignment, you can either (1) run the built-in secondary structure prediction (SSP) algorithm at this point or (2) import predictions generated from a third-party or web-based SSP method. Here we will import results generated using the PSI-PRED server [1]. Press the Import button and select FXa.psipass2. Prediction results will now be visible in the top window, where residues annotated with red letter Hs are predicted to form helices while residues annotated with blue letter Es are predicted to form beta sheets (Figure 6). Turn on the secondary structure information for the template sequence by pressing the View SSA icon (red cylinder) in the SPW toolbar; blue arrows indicate residues that form beta sheets while orange cylinders indicate residues that form alpha helices (Figure 6).

Figure 6. Visualizing secondary structure and conservation information during the Edit Alignment step of the SPW. Predictions for the target sequence are annotated as Hs (helices) or Es (beta sheets), while the secondary structure of the template is annotated as blue arrows (beta sheets) and orange cylinders (alpha helices). Strictly conserved aligned sequence positions are annotated

as a separate row in the alignment window.

8C. Identify strictly conserved regions based on homology. Press the Identify Globally Conserved Residues button; this will use protein family information from PFAM [2] to identify strictly conserved aligned positions. Conserved positions will be annotated in the sequence alignment (Figure 6).

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8D. Manually edit the alignment. Use the alignment slider icons to make manual changeds to the alignment so as to (a) ensure that globally conserved residues are aligned, (b) secondary structure elements are aligned to the greatest extent possible and (c) regions residing with secondary structure regions are gap-free. Use the edit SSP icon as necessary to edit the annotation of secondary structure regions as necessary. (Figure 7)

Figure 7. Manually edited alignment (yours may look slightly different).

When finished press Next. Note: if you have gaps in secondary struture regions of your template or query then an error message will appear. You can choose to ignore these gaps or try to fix them before moving on. If you have too many gaps in secondary structure regions then the model may fail to be built.

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8D. Build a model. Keep the default options and press the Build Model button. When the new model is built it will appear in the workspace (Figure 8). Note differences in the color coding of this model versus the one built using 1KIG as the template, in particular that much of the sequence is not conserved and loop insertions (red) are visible.

Figure 8. Resulting homology model of FXa built using human thrombin (PDB 2BVR) as the template. Sequence insertions are colored red.

9. Compare the two models to the structure of FXa. Open your project table. Include both models and the entry named 1F0R_prepped in the workspace. Color each ribbon uniquely by clicking and holding down the blue ribbon icon in the Representation toolbar and selecting Entry; the three structures should now be colored distinctly (Figure 9). Open the structural alignment panel (Tools -> Protein Structure Alignment in the Maestro menu bar); press Align. A window will pop up with the resulting structure-based sequence alignment and RMSD values and the structures will now be aligned in the workspace (Figure 9). Note that despite the low sequence identity between 1F0R and 2BVR, the structural model has a relatively low RMSD

Figure 9. Alignment of Factor Xa (green) to models built based on template structures

1KIG (blue) and 2BVR (peach).

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3. Assessing model quality

After building a model, it is always a good idea to examine its quality and fix any problems, such as steric clashes, bond length deviations and angles, etc, prior to using the model for tasks such as virtual screening. Most common structural problems can be fixed using the Protein Preparation Wizard.

10. Generate a protein quality report for the model based on 1KIG. Clear your workspace by pressing the Clear (broom) icon in the Workspace toolbar. Open the project table and include the model built based on 1KIG into the workspace. Display protein atoms by pressing the also display icon in the Display toolbar; choose Protein. Press the Protein Reports icon in the Proteins toolbar. A new panel will open that allows you to display various metrics associated with protein quality in table format (Figure 11). Double click on a row in the table to zoom into the corresponding region in the workspace, with the bond or atom highlighted in yellow (Figure 10).

Figure 10. Protein report for the model build on 1KIG (right side), and the corresponding workspace view of the highlighted entry in the table (left side). Use the drop down Display menu in the report to view other quality metrics.

11. View the Ramachadran Plot. Close the protein report and press the Ramachandran icon on the Proteins toolbar. In the Ramachandran plot that appears, you can visualize residues (represented by points) that reside within allowed (yellow), favored (reddishorange), or disallowed (white) regions of phi/psi space (Figure 11). Double click on a point to zoom into that residue in the workspace. Note that several residues are located outside of acceptable

phi-psi regions.

Figure 11. Ramachandran plot of the FXa mode built on 1KIG.

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4. Refining models The two most common tools for refining structures are the protein prep wizard and Prime loop refinement. Note that loop refinement is a CPU intensive calculation and should only be performed when deemed necessary.

12. Fix structural problems using the Protein Prep Wizard. Click on the Prep Wiz icon in the Project toolbar. In the panel that opens, click on the Refine tab (Figure 12). Under H-bond assignment, press Optimize. When the job finishes, press the Minimize button in the Restrained Minimization

section.

Figure 12. The refine tab of the Protein Preparation Wizard.

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Update the reliability report and Ramachandran plot; note that many of the problems have been fixed. You can use the Quick Torsion and Sculpt tools located in the Proteins toolbar to manually address any remaining problems.

13. Set up loop refinement for the model built on 2bvr. Clear the workspace. Open the project table and include in the workspace the entry for the model built on 2bvh. Navigate to the Prime loop refinement panel (Tasks -> Protein Refinement -> Refine Loops). In the window that opens, press the Non-Template button; the table should now be populated with loops that correspond to the red regions of the model (Figure 13). To include a loop(s) in the refinement, click in the box in the Run column. Press Run (Note: this calculation can take several hours to day depending on the number and on the length of the loops).

Figure 13. The loop refinement window.

References:[1] http://bioinf.cs.ucl.ac.uk/psipred/

[2] http://pfam.xfam.org/