Discovery Studio Tutorials - ndmctsgh.edu.t 1: Working with the mouse, the Sequence Window, and the...

Discovery Studio 1.5

Online tutorial

A tutorial helps you to increase your knowledge of Discovery Studio. The lessons are available for skill levels from beginning to advanced. This is a good place to start whether you are first learning Discovery Studio, or mastering a particular functionality within the software.

Some of the lessons require data files that can be downloaded here. Save the zip file to your computer and then extract the contents to a folder. To open any of the the data files, choose File | Open... from the menu bar within Discovery Studio, and then navigate to the location where the files were extracted.

Since Discovery Studio was released, the default RCSB web address for PDB file downloads has changed. To update the default setting in Discovery Studio, choose Edit | Preferences... from the menu bar to display the Preferences dialog. Within the Files Explorer, select PDB Location and change the Web Site option by selecting pdb.rcsb.org from the dropdown list. If you have a local PDB repository, Discovery Studio can instead download files from here. Change the PDB Location preferences, setting the Local File Path to the location of this repository. Depending on the structure, format, and compression scheme used within your repository, you may need to alter the Form of File preferences to match.

Lesson Description

Lesson 1 - Working with the mouse, the Sequence Window, and the 3D Window

Provides an introduction to mouse commands, display styles, their interactions with the Sequence and 3D Windows, and to aligning multiple protein structures.

Lesson 2 - Working with structural models Provides instruction for working with 3D pointers and text labels, calculating a Non-Crystallographic Symmetry (NCS) matrix, generating NCS mates, displaying crystal symmetry mates, and packing in a crystal lattice.

Lesson 3 - Building and editing small molecules Provides instruction for constructing small molecules useing a variety of tools and toolbars.

Lesson 4 - Docking ligands to a receptor and computing scores for the docked poses

Focuses on the computational methods that a computational chemist performs by docking a series of molecules, making assessments using various scoring functions, and analyzing the results.

Lesson 5 - Fitting single residues Provides instruction for using the residue-based fitting tools on individual residues to correct geometry, search side chain rotamers, mutate residues, and fit a residue to a density.

Lesson 6 - Homology modeling of an extracellular amylase protein

Focuses on the computational methods that a structural biologist performs, including protein sequence alignment, creating a model using the homology modeling method, and working with 3D models.

Standalone server documentation

The standalone server documentation provides instruction for installing and running the standalones by product. Choose from the following:

• CHARMm • DelPhi • Genfra • LigandFit • Ligand Scoring • Ludi • LudiScore • MODELLER

Lesson 1: Working with the mouse, the Sequence Window, and the 3D Window

Purpose: Provides an introduction to mouse commands and display styles, and their interactions with Discovery Studio Windows, and introduces multiple protein structure alignment.

Modules: Discovery Studio Visualizer

Time:

Prerequisites: None

Introduction to the 3D Window and the Sequence Windows

1. Start Discovery Studio

From the Windows Start menu, choose Programs | Accelrys Discovery Studio [version] | Discovery Studio.

If you have a Discovery Studio icon on your desktop, you can also start Discovery Studio by double-clicking this icon.

2. Open a 1TPO file

Choose File | Open... from the menu bar.

This displays the Open dialog.

On the Open dialog, navigate to and select the 1TPO.pdb file.

Note. Instructions for obtaining data files necessary to running this and other tutorials are available at http://www.accelrys.com/doc/life/dstudio/15.

This retrieves a beta trypsin file, 1TPO.pdb, from your local tutorial data file folder and opens it in the 3D

Structure View of the 3D Window.

Tip. Alternatively, if you have web access, choose File | Open URL... from the menu bar to display the Open URL dialog. Enter 1TPO (protein PDB identifier) in the Generate URL using the current PDB Location Preferences for PDB ID text box and then click the Open button.

3. Selecting objects

Note. The protein is surrounded by points representing water molecule oxygen atoms.

Double-click an oxygen atom (colored red by default) in a water molecule. Then double-click the same oxygen atom to select all components of the "chain" belonging to the water molecule.

All oxygen atoms change color to yellow to show that they are selected.

Choose View | Hierarchy from the menu bar.

This highlights one water chain in the Hierarchy View.

Choose View | Data Table from the menu bar and click the Chain tab within the Data Table View. Then CTRL + click the water rows (select the first cell in the first row and the first cell in the second row to highlight both the rows).

This selects both water chains in the Hierarchy and Data Table Views. All of the water oxygens change color to yellow on the protein structure to show they are selected.

4. Deleting water molecules

Ensure the 3D Structure View, Hierarchy View, or Data Table View is current by clicking in one of these views.

Delete the selected water oxygens. Choose Edit | Delete from the menu bar or press DELETE.

The selected water oxygens are deleted.

5. Exploring views

Try clicking the + and - signs in the Hierarchy View.

This allows you to explore the information available in the Hierarchy View by expanding and contracting the levels.

Note. You can drill down to the atom level and highlight specific groups (e.g., hydrophobic residues).

Try exploring the tabs in the Data Table View. Click the Molecule tab. Drag the Molecular Weight column in front of the Number of Atoms column. Click the AminoAcidChain tab and change the color of the chain.

Note. The white cells can be modified (e.g., Atom tab, element column) and the grayed cells cannot.

After running a protocol in Discovery Studio, job results sometimes populate this table by automatically adding additional column(s) of information. Note the useful information (e.g., the Partial Charge and Isotropic Displacement columns on the Atom tab).

Choose View | Data Table from the menu bar.

Unchecking this option closes the view and checking this option opens the view.

Mouse commands and display style functionality

3D structures can be manipulated in a variety of different ways using the buttons on the View toolbar. For a complete list of mouse commands and keyboard functions, see the Mouse and keyboard actions Discovery Studio 1.5 Help topic.

If the View toolbar is not displayed, as it is by default, choose View | Toolbars | View from the menu bar.

Check this command to display the toolbar. Unchecking this command hides the toolbar.

Tip. With the 3D Structure View current, hover your cursor over each button on the View toolbar to learn the button's function.You can perform simple manipulations of a structure in the 3D Structure View by clicking the Rotate, Zoom, and Translate tool buttons on this toolbar and then left-clicking and dragging in the 3D Structure View to cause an associated transformation of the view.

1. Tool buttons

Click each of the tool buttons associated with the following actions and drag the cursor around inside the 3D Structure View:

Select: Selects a region of the structure by left-clicking and dragging the lasso around a portion of the structure. Add to the selection by SHIFT + clicking and encircling another region with the lasso.

Rotate: Rotates the selected (highlighted) atoms by pressing CTRL in the rotate mode (but be careful because this may distort your structure undesirably). Use the SHIFT command while rotating the molecule to rotate in the Z plane. Rotate can also be utilized from the Translate or Zoom tool buttons while holding down the right mouse in these modes.

Translate: Translates the molecule in the XY plane by using left-click + drag or along the Z axis by using SHIFT + left-click and drag.

Zoom: Enlarges the view of the structure (zooms in) by dragging the cursor upward or to the right. Dragging the cursor downward or to the left decreases the structure's visual size (zooms out).

Tip. Other mouse tools are available when you display other toolbars (e.g., View | Toolbars | Sketching displays the torsion tool). Explore the use of the mouse to alter the view of the molecule and to make selections.

Click the Zoom tool button while dragging the cursor up and down.

This displays front and back clipping of the molecule.

Tip. If you have a wheel on your mouse, use CTRL + mouse wheel and SHIFT + mouse wheel

Click an atom to select the atom, double-click its parent residue to select the residue, and double-click an already-selected residue to select its parent chain.

This allows you to select different molecular hierarchical levels, progressively.

To deselect, click an object to make a new selection, or click in a blank area of the window to deselect everything.

2. Change the Display Style

Click the Select tool, right-click in the 3D Structure View, and choose Display Style... from the context window.

This displays the Display Style dialog.

Tip. Alternatively, to display the Display Style dialog when the 3D Structure View is current, you can press the CTRL + D in this view; choose View | Display Style... from the menu bar; or click the Display Style button on the View toolbar.

On the dialog, click the Atom tab, and from the Display Style control group, select Ball And Stick. Click the Protein tab, and from the Display Style control group, select Solid Ribbon. Click the OK button.

This applies your selected changes.

Note. You can change the coloring of atoms or residues in each tab based on different properties.

Working with the Sequence Window and the 3D Window

In this section, you will learn how the Sequence and 3D Windows can be used together.

1. Open the Sequence Window

Choose Sequence | Show Sequence from the menu bar to view the sequence for the molecule.

Note. Look at the two windows in this view to see the PDB name and the amino acid residues.

With the Sequence Window current, right-click the Sequence Window and choose Display Style... from the context menu.

This displays the Sequence Window Display Style dialog.

On the dialog, click the Residue Color tab, select Color By, and then select Secondary Structure from the dropdown list. Click the OK button.

This changes the residue colors based on secondary structure type.

Right-click in the Sequence Window, and choose Secondary Structure Cartoon from the context menu.

This displays the Kabsch-Sander secondary structure cartoon. The coloring of the residues should correspond to the secondary structure cartoon display. The blue arrows represent beta-strands, and the red, solid line represent alpha-helices.

Tip. There are multiple Sequence Windows for each new molecule. You can change the preference to open all sequences into one window. Choose Edit | Preferences... to display the Preferences dialog, and then, on the dialog choose Sequence Window | Add to Existing Window. Other preferences, including synchronize colors between sequence and structure windows as well as showing secondary structure, can be set on this dialog`.

2. Explore the mouse modes in the Sequence Window

Select a residue by clicking it and dragging over other residues with your cursor.

As you select residues, note the interactivity between the other views (e.g., the 3D Structure View and the Hierarchy View) and the selected residue name and number on the status bar.

Select a range of residues by dragging your cursor over the residues.

Add to the selection by SHIFT + clicking and dragging or dragging over additional residues.

Select the entire protein by clicking the sequence name in the Sequence Window.

3. Create the Catalytic Triad subset

Hover the cursor over any residue in the Sequence Window.

The residue ID is reported in the status bar.

Find and select the three residues of the Catalytic Triad - HIS57, ASP102, and SER195 (clicking and dragging over the residue adds it to the selection).

Tip. You can also use the Hierarchy View or Data Table View to select the residues.

Note. The numbers on the ruler in the Sequence Window run sequentially from 1 upwards. They do not reflect the residue numbering in the protein.

4. Create a group or subset

With whichever view you used to select the Catalytic Triad residues current, choose Edit | Group and enter Catalytic Triad and select Define. Choose View | Fit to Screen or click the

Fit to Screen button on the View toolbar.

The 3D Structure View zooms in on the selected group of residues.

Note. The group is added to the Hierarchy View (bottom of hierarchy) and Data Table View (group tab).

5. Alter the display style of the protein and the Catalytic Triad

With the 3D Structure View current and the Catalytic Triad still selected, deselect the triad by choosing Edit | Invert Selection from the menu bar. Choose View | Display Style... from the menu bar and, on the Atom tab of the 3D Structure View Display Style dialog, set the Display Style to None and click the OK button.

The entire protein is displayed as a ribbon with only the triad in ball and stick display style.

Aligning sequences in the Sequence Window and the 3D Window

In this section of the tutorial, you will work with the Sequence Window to manually superimpose sequences and then align the structures based on the sequence alignment.

1. Set a Default Display Style and load alpha-chymotrypsin 2CHA.pdb

Choose Edit | Preferences from the menu bar and choose the 3D Window page and then the Display Styles page of the Preferences dialog. Click the arrow on the Atom Display Style dropdown box and set the Display Style to Stick. Click the arrow on the Protein Display Style dropdown box and set the Display Style to Solid Ribbon. Click the OK button.

The default display style you just set will now be applied when you load alpha-chymotrypsin (2CHA.pdb).

Choose File | Insert from | URL... from the menu bar.

This displays the Open URL dialog.

Enter 2CHA (protein PDB identifier) in the Generate URL using the current PDB Location Preferences for PDB ID text box and then click the Open button.

Tip. You can change the location of the PDB by choosing Edit | Preferences | Files Explorer | PDB Location. This allows you to set the PDB to a local file path as well as change the form of the file (e.g,. compressed format .gz).

Tip. To see both molecules, choose View | Fit to Screen from the menu bar.

2. Show sequences in the same Sequence Window

Choose Sequence | Show Sequence from the menu bar.

This allows you to view the sequence for both molecules in a new Sequence Window.

Right-click in the Sequence Window and choose Secondary Structure Cartoon from the context menu.

This shows the secondary structure cartoon in the new Sequence Window.

Note. The status bar displays the % sequence identity and similarity. You can use this to monitor the alignment quality as you manually align the sequences.

3. Align the sequences manually for 1TPO and 2CHA

There are several techniques and tools to help you to judge sequence alignments. The % sequence identity and similarity and the residues' coloring are based on these properties.

Try coloring the sequences by sequence similarity by right-clicking in the Sequence Window and choosing Display Style... from the context menu. On the dialog, click the Background Color tab, select the Color By control, click the arrow on the dropdown, and choose Sequence Similarity from the list. Review the colors for the type of similarity and click the OK button.

Note. The sequences may already have been colored by similarity for this new window.

This colors positions in the alignment where residues are identical, or strongly and weakly similar.

Now manually align the sequences by inserting gaps pressing the SPACE bar to create them and pressing BACKSPACE to remove them. Add 11 spaces at the beginning (N-terminus) of the 1TPO sequence while watching the % sequence similarity change to 33.3% and noting the color change of the residue background.

This aligns the HIS57 Catalytic Triad residues of each protein.

4. Experiment with the layout of the Sequence Window

Right-click in the Sequence Window and choose Display Style... from the context menu. On the dialog, click the Display tab and set the font in the Font control. Right-click on the Sequence Window to display the context menu and check (to display) or uncheck (to hide) Wrapped View.

5. Superimpose the two proteins based on the sequence alignment

Select the 1TPO protein in the Hierarchy View and choose Structure | Superimpose | Superimpose by Sequence Alignment... from the menu bar.

In the Molecules to Superimpose field on the dialog, select 2CHA and click the OK button.

A text window displays the RMSD at 6.30 Angstroms over 222 residues.

Close this text window.

6. Color the protein 3D structure based on the sequence alignment coloring

Choose Edit | Preferences... from the menu bar.

This displays the Preferences dialog.

On the dialog, check the checkbox for Synchronize with 3D Window and Background Color to enable this option. Click the OK button.

This applies the coloring to the atoms.

In the Sequence Window, right-click and choose Display Style... from the context menu.

This displays the Sequence Window Display Style dialog.

On the dialog, click the Background Color tab, select the Color By option, click the arrow on the dropdown, and chose Sequence Similarity from the list. Click the Apply and then the OK button.

In the 3D Structure View, right-click and choose Display Style... from the context menu.

This displays the 3D Structure View Display Style dialog.

On the dialog, click the Protein tab, select the Color By option, click the arrow on the dropdown, and chose CAlpha.

Now the protein sequence and 3D structure coloring are synced.

Tip. Try adding spaces in the sequence to change the alignment and watch the coloring of the two views change.

This is the end of lesson 1.

Lesson 2: Working with structural models

Purpose: Provides an introduction to working with 3D pointers and 3D text labels, calculating a non-crystallographic symmetry (NCS) matrix and generating NCS mates, and displaying crystal symmetry mates and packing in a crystal lattice.


Time:

Prerequisites: None

Introduction

In this lesson, you will learn how to navigate a protein structure using 3D pointers and 3D text labels, and how to annotate a structure. Then, you will calculate a non-crystallographic symmetry (NCS) matrix for a protein based on specific structural segments and use this matrix to generate NCS mates, enabling you to build a complete molecular structure from a monomeric subunit. Finally, you will discover how to use the crystallographic symmetry tools in the Discovery Studio Visualizer to display crystal symmetry mates and packing in a crystal lattice.

This lesson covers:

• Part 1: Working with 3D pointers • Part 2: Using 3D annotations in 3D space • Part 3: Calculating an NCS matrix based on specified segments • Part 4: Build in NCS mates using a known NCS matrix • Part 5: Using crystallographic symmetry tools

Part 1: Working with 3D pointers

1. Start Discovery Studio



2. Open an insulin crystal structure



On the Open dialog, navigate to and select the 9INS.pdb file.


This retrieves an insulin crystal structure file, 9INS.pdb, from your local tutorial data file folder or from the

Protein Databank (PDB) on the web and opens it in the 3D Structure View of the 3D Window.

Tip. Alternatively, if you have web access, choose File | Open URL... from the menu bar to display the Open URL dialog. Enter 9INS (protein PDB identifier) in the Generate URL using the current PDB Location Preferences for PDB ID text box and then click the Open button.

Either press CTRL + H or choose View | Hierarchy from the menu bar.

The Hierarchy View is displayed.

3. Create and move a 3D pointer

Click the insulin molecule's 3D Structure View to make it the active document. In the Tools Explorer window, double-click the X-ray tool panel to display the X-ray tools.

Tip. Alternately, click the options arrow on the X-ray tool panel header and choose Restore from the drop-down menu.

Note. If the Tools Explorer is not visible, select View | Explorers | Tools from the menu bar to display it.

Select the Display Pointer command from the 3D Pointer tool group on the X-ray tool panel.

A diamond-shaped pointer appears in the center of the 3D Structure View.

Click the 3D pointer to select it.

Tip. Alternatively, click on the <PointCursor> item in the Hierarchy View to select the 3D pointer. Press the TAB key to change the focus from the Hierarchy View back to the 3D Structure View.

Click the Translate button on the View toolbar to enter translation mode. While holding down the CTRL key, left-click and drag the mouse within the 3D Structure View window.

This moves the pointer in the 3D Structure View to a position other than the center.

4. Recenter the 3D Structure View at the pointer position

In the Tools Explorer, click the Go To Pointer command in the 3D Pointer tool group on the X-ray tool panel.

The pointer becomes the center of the 3D View. This recenters the 3D Structure View at the pointer position.

5. Select the placement of the 3D pointer

Click any atom in the structure. Select the Place Pointer at Selection command from the 3D Pointer tool group on the X-ray tool panel.

The pointer is placed directly over the selected atom.

In the Hierarchy View, select the residue CYS20 from chain A. Click the Place Pointer at Selection command in the 3D Pointer tool group.

The pointer moves directly over the selected residue. The whole residue is selected and marked as the current residue.

6. Navigate through the structure

Click the Next Residue command in the 3D Pointer tool group on the X-ray tool panel.

The pointer moves directly over the Cα atom of the next residue (ASN21), navigating through the structure. The whole ASN21 residue is selected and marked as the current residue.

Click Next Residue again.

The pointer moves directly over the Cα atom of the next residue (PHE1), which is the first residue of the B chain. The PHE1 whole residue is now selected and becomes the current residue.

Click the Previous Residue command in the 3D Pointer tool group.

The pointer moves back to the Cα atom of the previous residue, ASN21 of chain A.

The Next Residue and Previous Residue commands make it easy to navigate through the protein chain while adjusting the view. This is particularly useful during X-ray crystallographic model building.

7. Hide or display the 3D pointer

Click the 3D pointer in the 3D Structure View or the Hierarchy View to select it. Choose View | Visibility | Hide from the menu bar.

The 3D pointer object located at the Cα atom of ASN21 disappears from the 3D Structure View.

Choose View | Visibility | Show from the menu bar.

The 3D pointer reappears.

8. Save the view with the current pointer object

The structure with the 3D pointer in its current location can be saved in an .msv file.

Choose File | Save As... from the menu bar to display the Save As dialog. Select Viewer Files (*.msv) from the Files of type drop-down list and enter 9ins_pointer as the File name. Select a location on your machine for the file and click the Save button.

The file 9ins_pointer.msv is created in the local folder that you specified. This file can be opened in

Discovery Studio and contains the 3D pointer object that you created and positioned.

9. Delete the 3D pointer object

Click the Remove Pointer command in the 3D Pointer tool group on the X-ray tool panel.

The 3D pointer is removed from the 3D Structure View and is deleted from the system.

Part 2: Using 3D annotations in 3D space

In this section, you will learn how to use 3D text labels to annotate and navigate through structures.

1. Create a 3D text label at a residue

Under chain A in the Hierarchy View, select CYS6. Click the Place Pointer at Selection command in the 3D Pointer tool group on the X-ray tool panel.

The center of the 3D Structure View moves to the Cα atom of CYS(A6) and a 3D pointer appears over this atom. The whole residue is selected.

Click the Add Label... command in the 3D Labels tool group.

Note. You might have to scroll down the X-ray tool panel to reach the 3D Labels tool group. Click in the X-ray tool panel and press PAGE DOWN or, if you have a wheel mouse, use the mouse wheel to scroll through the commands on the tool panel.

On the Add Text at 3D Pointer dialog, enter CYS(A6) in the Label Text text box and change the font to Times New Roman. Click the OK button.

A 3D text label appears at a position near the Cα atom of CYS(A6).

2. Create a 3D text label near a disulfide bridge

In the 3D Structure View, click the γ-sulphur atom, Sγ, of CYS(A6). Click the Place Pointer at Selection command in the 3D Pointer tool group on the X-ray tool panel.

The 3D pointer moves to the center of the γ-sulphur atom.

Click the 3D pointer in the 3D Structure View or the Hierarchy View to select it. Click the Add Label... command in the 3D Labels tool group.

This displays the Add Text at 3D Pointer dialog.

Enter Intrachain Disulfide in the Label Text text box and click the OK button.

A 3D text label is created near the disulfide bridge.

3. Edit a 3D text label

Select the 3D text label you just created near the disulfide bridge and click the Edit Label... command in the 3D Labels tool group on the X-ray tool panel to display the Edit Text dialog.

Click the Color chooser and select a color from the color palette that is displayed. Click the OK button to close the color palette. Click OK on the Edit Text dialog.

The color of the 3D text label is changed.

4. Create another label

Select ASN21 under chain A in the Hierarchy View and click the Place Pointer at Selection command in the 3D Pointer tool group on the X-ray tool panel.

The 3D pointer and 3D Structure View are now centered on the Cα atom of ASN(A21).

Click the Add Label... command in the 3D Labels tool group to display the Add Text at 3D Pointer dialog.

Enter C Terminus of Chain A in the Label Text text box and click the OK button.

A second text label is created and appears selected at the pointer position.

5. Navigate using the 3D text labels

With the most recently created label highlighted, click the Previous Label command in the 3D Labels tool group on the X-ray tool panel.

This recenters the view and moves the 3D pointer to the position of the previous 3D text label in the list, which, in this case, is Intrachain Disulfide.

Click the Next Label command in the 3D Labels tool group.

This brings the view and the 3D pointer back to the next 3D text label, C Terminus of Chain A in this case.

Select the label CYS(A6) and click the Go To Label command in the 3D Labels tool group.

The 3D pointer and the center of view move to the position of the CYS(A6) text label.

6. Delete a 3D text label

With the 3D text label CYS(A6) highlighted, press DELETE.

The 3D text label is removed from the 3D Structure View and is deleted from the system. You can also delete selected labels by choosing Edit | Delete from the menu bar.

7. Save the remaining 3D text labels with the molecular structure

Choose File | Save As... from the menu bar to display the Save As dialog. Select Viewer Files (*.msv) from the Files of type drop-down list and enter 9ins_3Dtext as the File name. Select a location on your machine for the file and click the Save button.

The structure with the 3D text labels you created is saved in an .msv file in the local folder that you specified.

This file can be opened in Discovery Studio.

Part 3: Calculating an NCS matrix based on specified segments

In this section, you will learn how to calculate a non-crystallographic symmetry (NCS) matrix based on specified structural segments.

In the crystallographic model building process, when a monomeric subunit of a molecule with NCS is completely built, you can use the NCS matrix and complete the molecular structure by generating NCS mates. In this and the following section, you will use tetrameric insulin as an example. This molecule contains two heterodimers, chain A and chain B, and chain C and chain D. The two dimers are related by an NCS. Chains C and D are the NCS mates of chains A and B, respectively. First, you will construct a hypothetical incomplete molecular model that contains one complete heterodimer (chains A and B) and a terminal loop containing a helical segment of chain D (D9 to D20). Then, you will calculate an NCS matrix based on this segment and its equivalent in chain B. In Part 4, you will use this NCS matrix to generate all NCS mates to complete the whole tetrameric insulin molecule.

1. Open a tetrameric insulin structure file

Close the currently open 3D window. Choose File | Open... from the menu bar to display the Open dialog. Navigate to and select 1PID.pdb file. Instructions for obtaining this file are available at http://www.accelrys.com/doc/life/dstudio/15. Click the Open button.

This opens a tetrameric insulin structure file, 1PID.pdb, in the 3D Structure View of the 3D Window.

Select Sequence | Show Sequence from the menu bar.

This displays the sequence of 1PID in a Sequence Window.

2. Prepare a hypothetical incomplete tetrameric insulin model

In the following steps, you will remove all water molecules, all residues of chain C, and all residues except for the terminal loop between residue 9 and 25 of chain D. The resulting hypothetical model will contain chain A, chain B, and the terminal loop that contains the helical segment between D9 and D20.

In the Hierarchy View, click Water to select all water molecules. Press DELETE.

The chain of water molecules disappears.

Click chain C in the Hierarchy View to select it. Press DELETE.

Chain C is removed from the structure.

Click to expand the details of chain D. Click the first residue, PHE1, in chain D. Hold down SHIFT while clicking on the residue GLY8 in chain D. Press DELETE.

You have created a hypothetical incomplete tetrameric insulin model containing chains A and B and a structure segment of chain D (residue 9 through 25) that you will use for this section and the next one in this lesson.

3. Prepare the equivalent segments to be superimposed

Because chains C and D are the NCS mates of chains A and B, specify the helical segment of B9-B20 as the equivalent segment of D9-D20.

Choose Structure | Superimpose | Superimpose by Residue... from the menu bar to display the Superimpose by Residue dialog.

Because all the segments belong to the same molecule, 1PID is automatically selected as both the Reference Molecule and the Molecule to Superimpose on the Superimpose by Residue dialog.

Note. When working with two separate molecules, the reference molecule must be identified by selecting it in the Hierarchy View before opening the Superimpose by Residue dialog.

On the Superimpose by Residue dialog, click on the cell in the Reference Chain column of the Matching Residue Ranges grid view and select D from the drop-down list. Select SER9 from the drop-down list as the Reference Start Residue. Select GLY20 as the Reference End Residue, B as the Mover Chain, and SER9 as the Mover Start Residue.

This adds the two equivalent segments of chains B and D into the system for superposition. Further equivalent segments could be added by clicking the Add button to create a new row in the Matching Residue Ranges grid view and specifying the chains and residues involved in the appropriate cells.

4. Generate an NCS matrix based on the equivalent segments

In the text box next to the Generate Transformation Matrix Only control, replace the default entry, NCS, with B_to_D_Ins. Click the OK button.

An NCS matrix named B_to_D_Ins and based on the equivalent segments that you specified is generated and stored. The Cα RMSD value of the segments (if they were to be superimposed) is output in a text window.

Close the text window where the RMSD value is reported

Part 4: Building in NCS mates using a known NCS matrix

In this section, you will learn how to generate the NCS mates to complete the molecular model of tetrameric insulin, based on the NCS matrix you calculated in Part 3.

1. Delete the remaining residues of chain D from the system

Click chain D in the Hierarchy View and press DELETE.

All of the remaining residues of chain D are deleted from the system.

2. Check the stored NCS matrix

Choose Structure | Superimpose | Edit Transformation Matrix... from the menu bar.

The Edit Transformation Matrix dialog is displayed. All existing matrices previously calculated or entered are available from the Transformation Matrix drop-down list, verifying that they have been saved.

Select B_to_D_Ins from the Transformation Matrix drop-down list. Click the OK button.

Now all the matrix elements, both the rotation matrix and the translation vector, are listed. You can manually edit each element and save the change to the matrix. However, for this lesson, no change is required.

3. Generate NCS mates

Click chain A in the Hierarchy View, then, hold down CTRL and click chain B to select both chains A and B.

Choose Structure | Superimpose | Apply Transformation Matrix... from the menu bar to display the Apply Transformation Matrix dialog.

Select B_to_D_Ins from the Transformation Matrix drop-down list. Ensure that the Selected Atoms Only checkbox is checked and check the Create Copy checkbox. Click the OK button.

Two additional chains, C and D, are added to the 3D Structure View and the Hierarchy View. The whole tetrameric insulin model based on the calculated NCS matrix is now constructed and completed.

Part 5: Using crystallographic symmetry tools

In this section, you will learn how to display crystal symmetry mates in the 3D Structure View and how to display packing in a crystal lattice.

1. Open an insulin crystal structure

Choose File | Open... from the menu bar to display the Open dialog. Navigate to and select 9ins.pdb file. Instructions for obtaining this file are available at http://www.accelrys.com/doc/life/dstudio/15.

Click the Open button.

This opens an insulin crystal structure file, 9INS.pdb, in the 3D Structure View of the 3D Window.

2. Display packing within a unit cell

Right-click in the 3D Structure View and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog.

Click the Cell tab. Ensure that the Line display option is selected and that the Label Axes checkbox is checked. Click the Color chooser and select white from the color palette that is displayed. Click the OK button to close the color palette. Click OK on the 3D Structure View Display Style dialog.

In the 3D Structure View, a unit cell box with marked cell axes is drawn in white.

Note. You may need to adjust the view using the tools on the View toolbar so that the entire unit cell is visible within the 3D Structure View.

Choose Structure | Crystal Cell | Edit Parameters... from the menu bar.

The Crystal Builder dialog is displayed, showing the unit cell parameters of this particular insulin crystal structure. You can use this dialog to make adjustments to these parameters, if required. For the purposes of this lesson, however, you should leave the unit cell parameters unchanged.

Click the Preferences tab. Select One Cell from the Symmetry Style drop-down list. Make sure that A, B, and C are set to 1 in the View Range controls and click the OK button.

All the atoms within a single unit cell of the crystal structure are displayed in the 3D Structure View.

Right-click in the 3D Structure View and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog. Click the Atom tab and select the Color By option. Choose Molecule from the drop-down list and click the OK button.

The symmetry copies of the molecules are drawn in different colors.

3. Dynamically update all symmetry copies of the residue being edited

In the Hierarchy View, expand the list of residues in chain B of the first 9INS molecule. Select PHE1 from the list and click the Place Pointer at Selection command in the 3D Pointer tool group on the X-ray tool panel.

The 3D Structure View is now recentered and zoomed in on the phenylalanine (B1) residue. Notice that two symmetry-related copies of PHE(B1) are displayed and are related in three-fold symmetry.

Translate the structure in the 3D Structure View by holding down the middle mouse button or wheel and dragging so that the view is centered mid-way between the three symmetry-related phenylalanines. Right-click on the toolbar to display a list of available toolbars. Ensure that visibility of the Sketching toolbar is toggled on.

If the Sketching toolbar was not previously available, it will be displayed with the other toolbars at the top of the screen.

Click the Torsion button on the Sketching toolbar. Press and hold the left mouse button while dragging the bond between Cα and the side chain of PHE(B1) in the original molecule.

The side chains of PHE(B1) in all three symmetry-related residues simultaneously rotate around the chi1 torsion angle.

4. Create a Cα representation of crystal packing

Make sure that no atoms or bonds are selected by clicking an empty area of the 3D Structure View. Right-click in the 3D Structure View and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog.

On the dialog, click the Atom tab and select the None option from the Display Style controls.

Click the Protein tab and select the Ca Wire display style option. In the Coloring controls, select the Color By option and choose Molecule from the drop-down list. Click the OK button.

The 3D Structure View now shows the Cα representation of the crystal packing of the insulin molecules within a unit cell. Each molecule is represented by a different color, as before.


Lesson 3: Building and editing small molecules

Purpose: Introduces the sketching tools.


Time:

Prerequisites: None

Introduction

In this lesson, you construct a small molecule using a variety of tools from the Sketching and Chemistry toolbars. The molecule you will build is the 1,4-benzodiazepine drug diazepam (Valium):

Molecular structure of diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one)

This lesson covers:

• Starting Discovery Studio • Opening a new 3D Window • Building the six-membered aromatic ring of the fused ring system

• Building the seven-membered ring • Adding the side chains • Changing element types • Adding the carbonyl group • Adding the final double bond • Adding hydrogen atoms and cleaning the structure

1. Starting Discovery Studio



2. Opening a new 3D Window

Select File | New | 3D Window from the menu bar.

A new 3D Window is displayed in the workspace.

3. Building the six-membered aromatic ring of the fused ring system

You will begin by sketching using carbon atoms only, you will then go back and change some of the carbon atoms to other element types, as appropriate.

Select Edit | Preferences... from the menu bar to display the Preferences dialog. Choose 3D Window | Sketch and Clean from the tree view on the left side of the dialog to display the Sketch and Clean subpage. Make sure that the Add And Update Hydrogens checkbox in the When Sketching section is unchecked. Close the Preferences dialog.

Select View | Toolbars | Chemistry from the menu bar to turn on the Chemistry toolbar, and then View | Toolbars| Sketching to turn on the Sketching toolbar.

Choose the Ring tool on the Sketching toolbar. Click in the center of the 3D Window.

A six-membered ring of carbon atoms is generated.

Choose the Select tool on the View toolbar and double-click anywhere on the ring to select all the atoms. Now click the Aromatic Bond button on the Chemistry toolbar to convert the single bonds in the ring to aromatic bonds.

You have now generated a benzene ring (minus the hydrogen atoms). The ring is shown in the resonant representation; the dotted lines indicate aromatic bonds.

Tip. The Ring tool generates a ring of carbon atoms connected by single bonds by default. However, if you hold down the CTRL key when you click in the 3D Window with the Ring tool, you can create an aromatic ring directly.

4. Building the seven-membered ring

Choose the Ring tool on the Sketching toolbar. Click and hold the mouse on one of the bonds of the six-membered ring. Drag the mouse until the display shows a ring size of seven atoms. Release the mouse.

Tip. Use the mouse wheel, if you have a wheel mouse, or press CTRL + + on the numeric keypad to zoom

in on the bond so that you can select it accurately. Click the Undo button on the Standard toolbar or press CTRL + Z to undo any mistakes. Continue clicking or pressing to undo multiple steps.

A seven-membered unsaturated carbon ring is fused to one of the bonds of the six-membered aromatic ring.

5. Adding the side chains

Now that you have sketched the basic core structure of diazepam, you need to add the second aromatic ring, the methyl group, and the chlorine substituent.

Choose the Sketch tool on the Sketching toolbar.

The general sketching tool allows you to sketch atoms and bonds freehand. The Sketch tool always sketches with carbon atoms.

On the seven-membered ring, click one of the carbon atoms that is adjacent to the fused bond (at the 5-position) and drag the cursor away from the atom, extending the indicator line to its fullest extent. Double-click to place the new carbon atom and terminate the chain.

Note. The indicator line shows you where the new atom will be created. The fullest extent of the line is set to the standard bond length for a carbon-carbon single bond.

A single carbon atom is attached to the seven-membered ring in the 5-position. Notice that a bond is automatically added from the ring to the newly sketched atom.

Choose the Ring tool on the Sketching toolbar. While holding down the CTRL key, click the new atom you just added.

A six-membered aromatic ring sprouts from the single-atom side chain attached to the seven-membered ring.

Choose the Sketch tool on the Sketching toolbar. On the seven-membered ring, click the other atom that is adjacent to the fused bond in the 1-position, drag the cursor away, and click again to place the new atom. Press ESC to terminate the side chain. Repeat this process to add another exocyclic carbon atom in the 7-position.

The basic atomic framework of diazepam is now complete, apart from the carbonyl group and the double bond in the diazepine ring; however, all the atoms are carbon, whereas diazepam contains two nitrogens and a chlorine atom.

6. Changing element types

Choose the Select tool on the View toolbar and click the 1-position atom in the seven-membered ring that has a single-carbon side chain attached to it.

This atom should be a nitrogen atom.

With the atom selected, press N on the keyboard.

The atom is colored blue, signifying that it is now a nitrogen atom.

Likewise, click the single carbon atom attached to the fused six-membered aromatic ring (at the 7-position) to select it.

This atom should be a chlorine atom.

Many of the elements that have abbreviations consisting of a single symbol have been assigned keyboard shortcuts for sketching (i.e., H, B, C, N, O, F, P, S, and I); however, elements with two-character abbreviations, like chlorine, do not have keyboard shortcuts assigned.

With the atom selected, right-click and select Element | Cl from the context menu.

The atom is colored bright green, signifying that it is now a chlorine atom.

The Element submenu provides easy access to the elements most commonly found in organic molecules. The same functionality can also be accessed by selecting Chemistry | Element from the menu bar.

Select the atom in the seven-membered ring bearing the phenyl substituent. Select Chemistry | Element | Table... from the menu bar to display the Change Element dialog.

The Change Element dialog allows you to select any element from the periodic table.

Click N in the periodic table on the Change Element dialog and then click OK.

The selected atom is changed to a nitrogen atom.

7. Adding the carbonyl group

One substituent remains to be added to your molecule, the carbonyl group.

Choose the Sketch tool on the Sketching toolbar. On the seven-membered ring, click the carbon atom in the 2-position (i.e., the one that is next to the nitrogen atom with the methyl substituent, but that is not part of the fused bond). Drag the indicator line outside the ring until it stops and click to create the new atom. Do not terminate the side chain yet.

A single atom is attached to the seven-membered ring. The sketching tool remains active, as shown by the fact that the indicator line remains visible. You could go on clicking to draw an aliphatic side chain, however, in this case, a single atom is all that is required.

With the indicator line still attached to the new atom, drag the cursor back to the ring carbon and click to create a double bond.

The single bond becomes a double bond.

Note. After this operation, the previously created atom is still selected, making it easy to change the element type.

Press O on the keyboard to change the selected carbon to an oxygen atom.

A keto group is added at the 2-position of the diazepine ring.

8. Adding the final double bond

Now you will change one of the single bonds in the seven-membered ring to a double bond.

Continuing to use the Sketch tool, click the C-N bond linking the 4- and 5-positions in the seven-membered ring.

The bond becomes a double bond.

Tip. There are several other ways you can create a double bond. With the bond selected, either click the Double Bond button on the Chemistry toolbar or press 2 on the keyboard. Alternatively, select the bond and then either choose Chemistry | Bond | Double from the menu bar or right-click and select Bond | Double from the context menu.

9. Adding hydrogen atoms and cleaning the structure

The basic structure of diazepam is now complete. All that remains is for you to add hydrogen atoms in appropriate positions and correct any anomalous bond lengths or angles.

Choose the Select tool on the View toolbar. If the double bond is still selected from the previous step, deselect it by clicking a blank area of the 3D Window. Click the Add Hydrogens button on the Chemistry toolbar.

Tip. Alternatively, select Chemistry | Hydrogens | Add from the menu bar.

Hydrogens are added to all the atoms in the structure containing unfilled valences based upon the common oxidation state of the atom, its hybridization, the formal charge on the atom, and the number and order of bonds to the atom. The geometry of the added hydrogens is determined by the orders of the existing bonds to the atom. All unfilled valences are filled with hydrogen atoms.

Often, the structure that results from a sketch may not be geometrically reasonable, for example, bond lengths and angles can be inappropriate for the atoms involved.

Select Structure | Clean Geometry from the menu bar to perform a simple geometry optimization.

Tip. Alternatively, click the Clean Geometry button on the Chemistry toolbar.

The Clean Geometry tool rapidly optimizes the geometry of the structure, taking into account element types, bond orders, number of bonds, and valences.

Repeat the Clean Geometry operation several times until you see no further changes in the geometry.

You have just built a 3D model of a diazepam molecule. The geometry of the model provides a reasonable starting point for further calculations, e.g., minimization.


Lesson 4: Docking ligands to a receptor and computing scores for the docked poses

Purpose: Introduces setting up and running a docking protocol, docking a ligand, and analyzing the scores.

Modules: Discovery Studio

Time:

Prerequisites: None

Background

Introduction

This tutorial focuses on the computational methods that a computational chemist performs. This tutorial covers:

• Loading the protein receptor • Defining the receptor and searching it for binding sites • Running the Docking protocol • Analyzing the docking results • Minimizing the docked poses • Rescoring the minimized ligand poses • Analyzing the scored minimized poses • Running a consensus score protocol • Analyzing the Consensus Score results

1. Loading the protein receptor

Start Discovery Studio.

If you have a Discovery Studio icon on your desktop, you may start Discovery Studio by double-clicking this icon.

From the File Explorer, right-click a directory in which input and output files are saved when running a protocol. Click Set Default from the context menu.

This sets the default working directory. When a protocol is run, its input and output files are saved into this directory.




On the dialog, double-click the file pdb1kim_protH.msv in the Files Explorer.

This opens the file in a 3D Structure View. This protein has been already prepared for this lesson. Ligands and all crystallographic waters have been removed, and hydrogens have been added.

2. Defining the receptor and searching it for binding sites

Check to see if the Binding Site tool appears in the Tools panel. If the Binding Site tool is not displayed, activate its display by selecting View | Tool Panels | Binding Site from the menu bar. Now, double-click the Binding Site tool.

This displays the Binding Site Tool.

In the 3D Structure View, select any atom of the protein receptor by clicking it.

The selected atom is highlighted with a yellow square.

In the Binding Site tool, click Define Selected Molecule as Receptor.

This defines the protein molecule as the receptor. If a Hierarchy View is open, you see SBD_Receptor appear as a new Group icon. If the Hierarchy View is not open and you wish to see it, click View | Hierarchy from the menu bar.

Now, click Find Sites from Receptor Cavities.

This employs a cavity detection algorithm that identifies binding site cavities inside the protein receptor. In this case, nine sites are identified. The sites are sorted by size, and the largest site is displayed.

Note. The Binding Site tool may be used to browse the list of sites using the Next Site and Previous Site buttons. Each site may be modified using the Contract Binding Site, Expand Binding Site, and Delete Site Points buttons.

3. Running the Docking protocol

From the Protocol Explorer, under the Receptor-Ligand Interactions group, open the Docking protocol by double-clicking it.

This opens the Docking protocol in the Parameters Explorer.

In the Parameters Explorer, click the Parameter Value cell next to the Input Target Receptor, and click pdb1kim_ProtH:1kim_proth.

pdb1kim_ProtH:1kim_proth is now displayed in the cell. The Parameter Value for Input Binding Site should automatically update and show Site 1 as the selected binding site along with the number of points, volume in Angstroms cubed, and partition level of 1.

Note. The Input Target Receptor parameter lists all molecules in all 3D Windows using the syntax of "Window name:Molecule name" as potential target receptors.

Click the Parameter Value cell next to the Input Ligands parameter, and click the button at the far right of the cell.

This opens the Specify Ligands dialog.

Click the All ligands from a file toggle and click the button.

This opens the Choose a ligands file dialog.

Double-click the file TK_xray_ligs.sd.

This selects the file and populates the name into the Specify Ligands dialog.

Click OK to close the Specify Ligands dialog.

The specified SD file is now entered into the Parameter Value cell for the Input Ligands parameter of the Docking protocol.

Click the Parameter Value cell next to the Energy Grid Forcefield parameter and select PLP1.

This specifies PLP1 as the energy function for docking.

Note. The simple and short-range nature of this function allows for faster docking runs compared with using Dreiding or CFF, so it has been chosen for use in this tutorial.

Click the Parameter Value cell next to the Conformation search Number of Monte Carlo Trials parameter. Clear the text in the cell and enter the value 5000.

This specifies a fixed value of 5000 Monte Carlo trials to sample for each ligand for the conformational searching step.

Note. The default entry for this parameter specifies the number of steps as a function of the number of ligand torsions resulting in longer searches for more flexible ligands. The choice of 5000 enables the tutorial to run somewhat faster without significantly degrading the quality of the results for the purpose of this tutorial.

Click the Parameter Value cell next to the Pose saving Maximum Poses retained parameter. Enter the value 5.

This specifies a value of up to 5 poses to save for each ligand. Only poses that are distinct based on RMS and energy criteria are saved.

Click the Parameter Value cell next to the Scoring Scores parameter. In the popup list that appears, check the scores for LigScore2, PLP1, PMF, Jain, and Ludi Energy Estimate 3.

The specified scoring functions are calculated for each docked ligand pose.

From the Parameters Explorer, click the Run button.

This runs the Docking protocol. This job typically takes several minutes to complete. The status of the job can be monitored in the Jobs Explorer.

4. Analyzing the docking results

After the job is complete, click the Start Date column in the Jobs Explorer, and ensure that the column is sorted in descending order such that the most recent job appears at the top. If the arrow is pointing upward, click again so the arrow is pointing down. Double-click any cell in the row corresponding to the Docking run. If you have not run any other jobs after submitting the docking run, this should be the first row in the Jobs Explorer.

This opens the Files Explorer to the Output folder of the Docking job.

Double-click the BrowseMolecules.pl file in the Output folder.

This simultaneously opens the resulting docked ligand poses of the Docking job into a Table Browser, and opens an associated 3D View containing the first docked ligand pose and the protein receptor used for the calculation.

Right-click any cell of the Table Browser, and select the Group By... command.

This opens the Group By dialog.

In the popup list under Select column to group by:, scroll down and select MOL_NUMBER. Click OK to close the dialog.

The Table Browser is now split into two views. The left view contains a list of all ligands, and the right view contains a list of all poses for the first ligand.

Click any cell in the right Table Browser. Now use the up and down arrow keys to scroll through the list of poses.

Observe that the docked pose changes in the 3D View.

Select another row in the left Table Browser

Observe that the 3D View now displays the first pose of the selected ligand, and the right Table Browser now contains a list of poses for the selected ligand.

Right-click in the left Table Browser, and use the Group By... command to return the Group By selection to <None>.

The split view of the Table Browser disappears and all poses of all ligands are now shown in the single Table Browser.

Click the column heading labeled -PLP1. Now, with the CTRL key depressed, also click the column labeled LigScore2_Dreiding.

Both columns are highlighted in the Table Browser.

Choose Chart | Simple Point Plot from the menu bar.

A point plot is generated with the -PLP1 score plotted on the X-axis and the LigScore2_Dreiding score on the Y-axis for all the ligands and poses. A number of the LigScore2 values are negative. This is because the PLP1 docking function has a very soft repulsive core, whereas LigScore2 tends to strongly penalize poses forming bumps with the receptor.

In the plot, use the mouse to select the lowest point on the plot by circling the mouse cursor around the point with the left mouse button depressed.

The point is highlighted in yellow.

Return to the docked - Table Browser window and scroll the table down to row 19.

This row is now highlighted corresponding to the point picked in the plot. The value of the LigScore2_Dreiding cell in this row is approximately -13.5. The ligand pose is now displayed in the 3D View.

In the 3D View, right-click empty space and select Receptor-Ligand Bumps from the context menu.

Short contacts between the ligand pose and the protein receptor are displayed in magenta.

5. Minimizing the docked poses

In the Protocols Explorer, double-click In-Situ Ligand Minimization under the Receptor-Ligand Interactions group.

The In-Situ Ligand Minimization protocol opens in the Parameters Explorer.

In the cell next to Input Receptor, select pdb1kim_ProtH:1kim_proth.

Note. If you have previously closed the pdk1kim_ProtH window, please re-open this molecule into a new 3D Window as described above in Section 1 under Load the protein receptor.

This selects the protein as the receptor molecule for the In-Situ Ligand Minimization protocol.

In the cell next to Input Ligand File, click the button at the far right of the cell.


Click the All ligands from a table browser toggle. docked should already be selected as the name of the Table Browser. Click OK to close the dialog.

The name docked appears in the cell next to Input Ligand File.

Accept all other defaults. Click the Run button to run the protocol.

The protocol runs, and a new row appears in the Jobs Explorer table. This protocol typically takes several minutes to complete.

After the run completes, the value Success appears in the Status column of the job in the Jobs Explorer. In the Jobs Explorer, double-click any cell of this job.

This opens up the Output folder for this job in the Files Explorer. Note where this folder resides in the folder hierarchy.

6. Rescoring the minimized ligand poses

In the Protocols Explorer, double-click the Scoring protocol.

The Scoring protocol opens in the Parameters Explorer.

In the cell next to Input Target Receptor, select pdb1kim_ProtH:1kim_proth.

pdb1kim_ProtH:1kim_proth is now displayed in the cell. The Parameter Value for Input Binding Site should automatically update and show Site 1 as the selected binding site along with the number of points, volume in Angstroms cubed, and partition level of 1.

Click the Parameter Value cell next to the Input Ligands parameter, and click the button at the far right of the cell.


Click the All ligands from a file toggle and click the button.

This opens the Choose a ligands file dialog.

Browse to the Output folder of the In-Situ Ligand Minimization job, and double-click the file minimized.sd.

This selects the file and populates the name into the Specify Ligands dialog.

Click OK to close the Specify Ligands dialog.

The specified SD file is now entered into the Parameter Value cell for the Input Ligands parameter of the Scoring protocol.

Click the Parameter Value cell next to the Scoring Scores parameter. In the popup list that appears, check on the scores for LigScore2, PLP1, PMF, Jain, and Ludi Energy Estimate 3.

The specified scoring functions are calculated for each docked ligand pose.


This runs the Scoring protocol. This job typically takes about a minute to complete. The status of the job can be monitored in the Jobs Explorer.

7. Analyzing the scored minimized poses

After the Status of the Scoring job is shown as Finished in the Jobs Explorer, double-click in the row corresponding to that job.

This opens the Files Explorer to the Output folder of the Scoring job.

Double-click the BrowseMolecules.pl file in the Output folder.

This simultaneously opens the scored ligand poses of the job into a Table Browser, and opens an associated 3D View containing the first ligand pose and the protein receptor used for the calculation.

Scroll down the Table Browser to row 19, which should correspond to MOL_NUMBER 4 and POSE_NUMBER 4. Use the column scroll bar to scroll over to the column with the LigScore2_Dreiding heading.

The LigScore2_Dreiding value for this pose is now approximately 4.7. Using the unminimized pose, the LigScore2_Dreiding value was approximately -13.5, so the minimization has significantly improved the LigScore2_Dreiding estimate for this pose.

Click the column heading labeled -PLP1. Now, with the CTRL key depressed, also click the column labeled LigScore2_Dreiding.

Both columns are highlighted in the Table Browser.

Choose Chart | Simple Point Plot from the menu bar.

A point plot is generated with the -PLP1 score plotted on the X-axis and the LigScore2_Dreiding score on the Y-axis for all the ligands and poses. There is a significantly improved correlation between the PLP1 and LigScore2_Dreiding values for the minimized poses compared with the plot previously generated using the unminimized poses from the Docking protocol.

8. Running a consensus score protocol

In the Protocols Explorer, double-click the Consensus Score protocol.

The Consensus Score protocol opens in the Parameters Explorer.

Click the Parameter Value cell next to the Source parameter, and click the button at the far right of the cell.

The Specify Ligands dialog opens.

Click the All ligands from a table browser toggle, and select scored as the name of the Table Browser. Click OK to close the dialog.

The name scored appears in the cell next to Source.

Click the Parameter Value cell next to the Scores for Consensus parameter.

The Scores for Consensus dialog opens with 2 listboxes displayed. The list of all available fields from the Table Browser available to choose for the Consensus Score calculation is shown in the listbox on the left. The listbox on the right is empty.

With the CTRL key depressed, select the following entries from the list: -PLP1, -PMF, Jain, LigScore2_Dreiding, and Ludi_3.

The selected entries are highlighted.

Click the >> button to transfer the selection to the right listbox. Click OK to close the dialog.

The selected scores are displayed in the cell next to the Scores for Consensus parameter in the Parameters Explorer.


This runs the Consensus Score protocol. This job typically takes less than a minute to complete. The status of the job can be monitored in the Jobs Explorer.

9. Analyzing the Consensus Score results

After the Status of the Consensus Score job is shown as Finished in the Jobs Explorer, double-click in the row corresponding to that job.

This opens the Files Explorer to the Output folder of the Consensus Score job.

Double-click the consensus.sd file in the Output folder.

This should open the file into a Table Browser labeled consensus.

Note. You may need to ensure that the File Types Preferences for sd files is set to open in a Table Browser. This can be done using the Edit | Preferences... command from the menu bar to open the Preferences dialog. Select the File Types preference in the hierarchy list located under the Files Explorer group. Ensure that the Window Type for sd files is set to Table Browser.

A new column has been added labeled Consensus.

Double-click twice on the Consensus column header to sort the table according to the Consensus score in a descending order (i.e. higher scores are at the top).

Observe that the first row has a Consensus score of 5 and corresponds to the 1e2k_lig molecule. Scrolling the table to the right shows that this entry corresponds to POSE_NUMBER 5 for this molecule. Examining the values of the scores for the 5 scoring functions used in the calculation shows they all generated a high score for this pose resulting in its Consensus score value of 5.


Lesson 5: Fitting single residues

Purpose: Introduces the X-ray and X-BUILD tools.


Time:

Prerequisites: None

Introduction

In this lesson, you use the residue-based fitting tools on individual residues to correct geometry, search side chain rotamers, mutate residues, and fit a residue to a density.

Then you learn use X-BUILD tools to edit the main chain atoms and fit them to the electron density. This lesson covers:

• Starting Discovery Studio • Loading 1DB1 molecule • Bringing up a Hierarchy View • Typing the molecule • Refining the residue PHE279 • Searching side chain rotamers for residue Thr280 • Mutating the residue valine to methionine • Refining a water molecule

• Edit and refine main chain atoms: • Starting Discovery Studio. • Loading 1DB1 molecule • Bringing up a Hierarchy View • Typing the molecule • Adjusting the peptide plane • Adjusting the omega angle • Refining the distorted main chain peptide plane by real-space refinement • Flipping the peptide plane by 180° • Moving the Cα atom while refining the whole residue in real space

1. Starting Discovery Studio



2. Loading 1DB1 molecule

Go to File | Open.... On the Open dialog, select 1db1_xbuild.msv and click Open.

In the 3D Window the structure of the human nuclear receptor appears (with a 2Fo-Fc map for real-space

refinement). A 3D Pointer is located at the Cα position of Phe279.

3. Bringing up a Hierarchy View

Click the 3D Window to make it the current window and type Ctrl + H to launch the Hierarchy View. Alternatively, make sure Hierarchy is checked on the View menu.

The Hierarchy View appears.

Click the + sign to the left of 1db1 to expand its contents.

This molecule contains three "chains":

A: one polypeptide chain <Chain>: a ligand residue Water: water molecules

4. Typing the molecule

Select Edit | Preferences and select the Typing page on the Preferences dialog. Set the Forcefield to XPROLIG and make sure Fix hydrogens is disabled. Enable the option Automatically re-type molecule when chemistry changes and click OK.

The molecule can now be typed according to the XPROLIG forcefield.

Click Chemistry | Type Molecule.

This molecule is now typed and ready for the following session.

5. Refining the residue PHE279

Click the 3D Window to make it the current window. To make sure no atom is selected, click a blank space

in the 3D View. Then, double-click the Cα atom where the 3D Pointer is located.

The PHE279 residue is selected. You can verify this is so by finding PHE279 under the A chain in the Hierarchy View.

Make sure Tools is checked under View | Explorers. Make sure X-ray is selected under View | Tool Panels

Click the Place Pointer at Selection button in X-ray Tools.

The residue, PHE279, is now centered in the 3D View and set as the current residue.

Note that the phenyl ring is distorted and the side chain is out of the density.

Make sure X-BUILD is selected under View | Tool Panels

On the X-BUILD tools panel, click the Regularize Selected Zone button.

After a moment, the phenylalanine side chain is regularized and the corrected side chain coordinates are updated.

Make sure PHE279 is still selected. If PHE279 is no longer selected, you can click it in the Hierarchy View or double-click one of its atoms to reselect it.

On the X-BUILD tools panel, click the Fit Side Chain by Real Space Refinement button.

This fits the sidechain within the density using the real space refinement method. Now the sidechain is mostly in the density.

On the X-BUILD tools panel, click the Refine One Residue button.

This further refines the whole residue into the density. You can rotate and translate the structure to confirm that the residue, PHE279, is now fit into the density.

On the X-BUILD tools panel, click the Regularize Selected Zone again to clean up the geometry.

Now, the PHE279 residue fits within the density with good geometry.

6. Searching side chain rotamers for residue Thr280

On the X-ray tools panel, click the Next Residue button.

THR280 is centered in the 3D View and selected.

As you can see, the THR280 side chain appears to be outside the density.

Select Edit | Preferences and select the X-BUILD page on the Preferences dialog. Click the Conformation tab. Make sure that Lovell, Word and Richardsons is set as the rotamer library. Click OK.

On the X-BUILD tools panel, click the Search Rotamers... button.

A dialog is displayed containing three rotamers, the probabilities of the rotamers in the library, and the corresponding Chi-1 angle values.

Within the Select Rotamer dialog, click the first rotamer.

The first rotamer appears in the 3D View. This rotamer does not fit into the density.

Click the second rotamer in the Select Rotamer table.

The second rotamer appears in the 3D View. The rotamer fits within the density and a clear hydrogen bond is made between the gamma oxygen OG1 of the residue THR280 and the delta oxygen OD2 of ASP282. This is the correct side chain conformation for THR280.

Click OK in the Select Rotamer dialog.

The coordinates of the last side chain rotamer are now updated.

7. Mutating the residue valine to methionine

On the X-ray tools panel, click the Next Residue button.

VAL281 is centered in the 3D View and selected.

As you can see, the density of the whole side chain pocket is not appropriate to this valine. Rather, the density looks more like a methionine.

On the X-ray tools panel, select Met under Mutate Amino Acid. Note that you might have to hit the Page Down (PgDn) button to see this tool.

In a few seconds, the methionine residue replaces valine at position 281. The residue is automatically fit into the density. The coordinates of the new residue are updated. The change of residue type is also updated in the Hierarchy and Sequence Views.

8. Refining a water molecule

On the Hierarchy View, click the + sign to the left of Water to expand its contents. Select HOH500 from the list.

On the X-ray tools panel, click the Place Pointer at Selection button. Note that you might have to hit the Page Up (PgUp) button to see this tool.

The selected water HOH500 is centered in the 3D View.

Using the right-mouse button, rotate the structure to see that this water is not positioned at the center of the density peak.

On the X-BUILD tools panel, click the Refine One Residue button.

The water molecule is quickly refined and moved to the center of the density.

Edit and refine main chain atoms

1. Starting Discovery Studio.

Note. If you have just completed the previous section, you can go directly to step 5.



2. Loading 1DB1 molecule

Go to File | Open.... On the Open dialog, select 1db1_xbuild.msv and click Open.

In the 3D Window the structure of the human nuclear receptor appears (with a 2Fo-Fc map for real-space

refinement). A 3D Pointer is located at the Cα position of Phe279.

3. Bringing up a Hierarchy View

Click the 3D Window to make it the current window and type Ctrl + H to launch the Hierarchy View. Alternatively, make sure Hierarchy is checked on the View menu.

The Hierarchy View appears.

Click the + sign to the left of 1db1 to expand its contents.

This molecule contains three "chains":

A: one polypeptide chain <Chain>: a ligand residue Water: water molecules

4. Typing the molecule

Select Edit | Preferences and select the Typing page on the Preferences dialog. Set the Forcefield to XPROLIG and make sure Fix hydrogens is disabled. Enable the option Automatically re-type molecule when chemistry changes and click OK.

The molecule can now be typed according to the XPROLIG forcefield.

Click Chemistry | Type Molecule.

This molecule is now typed and ready for the following session.

5. Adjusting the peptide plane

Click the + sign to the left of 1db1 to expand its contents, then click the + sign to the left of chain A to expand its contents. Select the residue ARG158. Click the Place Pointer at Selection command in X-ray Tools.

The residue, ARG158, is now centered in the 3D View and set as the current residue.

To make sure no atom is selected, click a blank space in the 3D View.

Note. In the following steps, it may be helpful to turn off the density in order to see what you are doing. To do so, in the Hierarchy View, click 1DB1_2fofc.map to select the density. Then select View | Display Style. On the Isosurface tab, select Off under Display Style and Click OK.

Make sure Tools is checked under View | Explorers. Make sure X-ray is selected under View | Tool Panels

Select the main chain carbonyl carbon of ARG158. Click the Place Pointer at Selection button in X-ray Tools. Then click the Go To Pointer command.

The 3D View is centered on the peptide plane connecting the residues ARG158 and VAL159.

Under View | Toolbars, make sure that the Sketching toolbar is checked. On the Sketching toolbar, select

the Torsion Tool .

CTRL + click and drag the peptide bond (C-N) of this peptide plane.

A green torsion monitor appears. When dragging the mouse, the peptide plane is rotated about an axis between the two Cαs of the peptide plane.

Drag this torsion angle until a value of approximately 115° is indicated.

Note. To turn the density display back on, in the Hierarchy View, click 1DB1_2fofc.map to select the density (the density is now invisible, but this step is still necessary). Then select View | Display Style. On the Isosurface tab, select Quad Mesh under Display Style and Click OK.

Now the peptide plane is outside the density.

6. Adjusting the omega angle

Drag the peptide bond (C-N) of the same peptide plane.

Note. Again, for clarity you may wish to turn off the density display, as described in the notes above.

When dragging the mouse, a plane defined by a four-atom subgroup (Cα, C, O and N) of the peptide plane is rotated about an axis between the two C of ARG158 and the N of VAL159.

Drag this torsion angle until a value of approximately 150° is indicated.

Now the peptide plane is distorted and is completely outside the density.

7. Refining the distorted main chain peptide plane by real-space refinement

The previous step generated a main chain with distorted geometry. This can happen sometimes during model building. You will use the XBUILD tool to correct the model and fit it to the density.

Make sure X-BUILD is selected under View | Tool Panels

Select the peptide bond between the C of ARG158 and the N of VAL159. On the X-BUILD tools panel, click the Fit Main Chain by Real Space Refinement button.

In a few seconds, the previously distorted peptide plane is refined and moved back to the density.

8. Flipping the peptide plane by 180°

Click the peptide bond (C-N) of the peptide plane described in steps 5 and 6. On the X-Ray tools panel, click the Flip Peptide button.

This automatically rotates the peptide plane 180° around the two neighboring Cα atoms.

Click the Flip Peptide button again.

This rotates the peptide plane back to its previous location.

9. Moving the Cα atom while refining the whole residue in real space

Sometimes the Cα atom is not well-positioned for model-building. An X-BUILD tool allows you to edit the Cα position while making sure all atoms of the residue are fitted in the density. Here you adjust the Cα atom of GLN152 as an example.

Click the + sign to the left of 1db1 to expand its contents, then click the + sign to the left of chain A to expand its contents.

Select the residue GLN152. Click the Place Pointer at Selection command in X-ray Tools. The residue, GLN152, is now centered in the 3D View and set as the current residue. As you can see, the C atom of GLN152 is slightly off the center of the density.

On the X-BUILD tools panel, click the Move Atom and Refine Residue button.

The side chain of GLN152 is first fitted to the density with the Cα atom fixed. The newly fitted residue is now colored white. The position and the color of the original conformation remain the same. Discovery Studio is now waiting for an atom to be selected and moved.

Unselect the residue GLN152 by clicking a blank spot in the 3D View. Then, click to select the Cα atom of GLN152.

The Cα atom is now selected.

On the View toolbar, click the Translate button. CTRL + drag the mouse slowly so that the Cα atom moves towards the center of density.

While the Cα atom is moved, all side chain torsion angles of the residue are simultaneously fitted to the density and the atom positions are interactively refreshed. Stop at a position where the Cα atom is reasonably placed.

Click the Move Atom and Refine Residue button again on the X-BUILD tools panel to accept the conformation.

The adjusted conformation replaces the original conformation.

10. Move the Cα atom while regularizing the geometry of a selected zone

Similarly, the Cα atom may be moved while the geometry of a selected zone of residues is simultaneously regularized. Here you again adjust the Cα atom of GLN152 as an example.

In the Hierarchy View, select three residues: CYS151, GLN152, and PHE153 (CTRL-click to select the group).

The three connecting residues are now selected.

Click the Move Atom and Regularize Zone button on the X-BUILD tools panel.

Discovery Studio is now waiting for an atom to be selected and moved.

Unselect the three residues by clicking a blank spot in the 3D View. Then, select the Cα atom of GLN152.

The Cα atom is now selected.

On the View toolbar, click the Translate button. CTRL + drag the mouse slowly to move the Cα atom.

While the Cα atom is moved, all three residues in the selected zone are simultaneously regularized and the atom positions are interactively refreshed. Stop at a position where the Cα atom is reasonably placed.

Click the Move Atom and Regularize Zone button again on the X-BUILD tools panel to accept the conformation.

You are prompted to Accept or Reject the conformation.

Click Accept.


Lesson 6: Homology modeling of an extracellular amylase protein

Purpose: Introduces the tools available for structural biologists in Discovery Studio and illustrates how these tools might be used in a typical drug development project.

Required Functionality (and Modules): Discovery Studio Visualizer Pro, BLAST (from Protein Similarity Search), Build Models (from MODELER), Multiple Sequence Alignment (from Protein Families), Verify Protein (from Protein Health), and Electrostatic Potential (as part of DelPhi from Biopolymer).

Time:

Prerequisites: installation of the Discovery Studio 1.5 client and server and installation of the BLAST databases (e.g., PDB) from the separate BLAST database DVD into the DS Modeling 1.5 server.

Background

Three computational scientists from the different disciplines (bioinformatics, structural biology, and medicinal chemistry) are working together on a project that will eventually lead to a synthetic drug candidate with the most promising properties for drug development.

The general workflow for the above project could be represented as follows.

• The bioinformatician uses Accelrys Gene to:

1. Analyze a newly discovered gene - an extracellular α-amylase gene that was recently cloned and sequenced from Aeromonas hydrophila (a proteobacteria).

2. Determine the gene's open reading frame. 3. Translate the gene to its protein form (443 amino acids). 4. Store the protein sequence in a sequence file.

• The structural biologist obtains the protein sequence (NCBI identifier P41131, gi:728848) from the saved sequence file and further characterizes the protein.

The structural biologist uses Discovery Studio to:

1. Perform a protein sequence similarity search, using the BLAST protocol, to determine a template (1BVN) for homology modeling.

2. Align the protein sequence with the homologous 1BVN sequence using the Multiple Sequence Alignment protocol and manual alignment adjustment.

Note. 1BVN (protein PDB identifier) is an experimental X-ray crystal structure of a pig pancreatic α-amylase protein complexed with a proteinaceous inhibitor called Tendamistat.

3. Create a 3D homology model using the Build Models protocol. 4. Analyze the 3D model using the Verify Protein and the Electrostatic Potential protocols and,

optionally, optimize or analyze it using the Simulation protocols and the Evolutionary Trace tools). 5. Determine, using the Visualization tools, the interface residues of the 1BVN α-amylase P

chain, which are very similar to the residues in the 3D model. The residues, which are the chemical features of the interface, are saved as subsets in the .msv file for the next step.

6. Compare the model and the 1BVN interface (using the Visualization tools). • The medicinal chemist uses Accelrys MedChem Explorer to:

1. Obtain the subsets from the saved .msv file.

2. Perform a chemical feature-based query. 3. Create a pharmacophore. 4. Compute ADME properties for the library molecules in order to focus in on the members

(drug candidates) with the most promising properties for drug development.

Introduction

This lesson focuses on the computational methods that the structural biologist uses in the example workflow above, aiming to introduce you to some of the protein modeling tools and protocols in Discovery Studio and to demonstrate how they might be used in a drug development environment.

This lesson covers:

• Starting Discovery Studio and preparing for experiments • Identifying a homologous structure using the BLAST protocol • Analyzing the BLAST results • Locating and opening the selected structure • Aligning the sequences using the Multiple Sequence Alignment protocol • Manually adjusting the alignment • Building a 3D model • Determining the binding interface residues • Evaluating the model • Assessing the validity of the 3D structure using the Verify Protein protocol • Calculating electrostatic potential using DelPhi (optional)

1. Starting Discovery Studio and preparing for experiments



In the Files Explorer, right-click the directory where you want to save the protocol files and choose Set Default from the context menu.

This sets a default working directory. The input and output files of the protocols will be saved into this folder automatically.

Choose File | Change Server... from the menu bar to display the Change Server dialog. Enter the name of the machine where the Discovery Studio server is installed and the number of the port to be used to access it in the Server name text box using the format <server name>:<port number>. If you installed the

server on the same machine as the Discovery Studio client, you should leave the default setting, localhost:9941, unchanged.

You have now set the server that you will use.



On the Open dialog, navigate to and select the virtual-pharma-seq.pir file.


This retrieves the file from your local tutorial data file folder and displays it in the Sequence Window.

2. Identifying a homologous structure using the BLAST protocol

In the Protocols Explorer, double-click the BLAST protocol under the Protein Modeling protocol group.

This displays the BLAST protocol in the Parameters Explorer.

In the Parameters Explorer, click the cell in the Parameter Value column for the Sequence parameter and select virtual-pharma-sequence:virtual-pharma-seq from the dropdown list.

The name of the Sequence Window is displayed as virtual-pharma-sequence and the name of the sequence is given as virtual-pharma-seq.

Click the cell in the Parameter Value column for the Database parameter and select PDB from the dropdown list.

This specifies that the PDB database is to be used for the BLAST search.

Note. The PDB sequence database is installed prior to running the protocol (refer to the installation instructions for details).

Note. The results you achieve from running the protocols in this lesson may change if you alter the default parameters or use a different or updated PDB database.

Review the other settings in the Parameters Explorer, but do not change the default parameters. Click the

Run button.

The BLAST run is started and the Jobs Explorer displays the status of the job as it progresses. When the protocol is complete, the BLAST report file is automatically opened in a graphical BLAST Results Window.

Note. Protocols are run on the Pipeline Pilot server component of Discovery Studio and so are separate from the client. This means that the job will run on the server (in the background if the Pipeline Pilot server is installed on the same machine as the client), leaving you free to work other files in the Discovery Studio Visualizer, to set up and launch other protocols on different servers, or even to exit Discovery Studio while you are waiting for the initial run to complete.

When the protocol is complete (which should take less than 20 seconds), the Job Completed dialog will be displayed, unless you have previously unchecked the Notify me when a job completes checkbox on the Job Completed dialog.

Click OK to close the Job Completed dialog and display the BLAST Results Window.

If the Job Completed dialog is not displayed, double-click the BLAST job in the Jobs Explorer to open the output folder from the BLAST job in the Files Explorer. Double-click the virtual-pharma-seq.xml file in the Output folder to display the BLAST Results Window.

The BLAST job folder is saved in the default folder that you specified in step 1 above. The BLAST folder (job) name is automatically generated by Discovery Studio, starting with BLAST and followed by a date stamp and a set of unique identification numbers. You can rename this folder by right-clicking the folder and choosing Rename.

3. Analyzing the BLAST results

In the BLAST Results Window, click the Table View tab at the bottom of the window.

The Table View shows the hits, with one line per sequence. Cells in grey cannot be edited, unlike some of the cells in the Data Table view of the 3D Window.

Note. The hits are initially ranked according to the E-value (the probability of a gapless alignment sequence occurring by chance alone), with the best, lowest value first.

In the BLAST Results Window, select the Map View tab.

The Map View shows the coverage of the hits in a map, with one line per sequence. The bars are colored according to the bit score of the hits (with above 400, red, being the best hits). The query sequence, virtual-pharma-seq in this case, is displayed at the top of the Map View represented as a line 465 residues long. Hovering the cursor over a hit on the Map View shows the description of the database sequence, the sequence accession id, the start and end position of the query sequence and the start and end of the database sequence of the hit, and the scores of the hit, the sequence length of the hit, and the scores (score, identities, positives and gaps). When a database sequence matches the query sequence in multiple regions (multiple hits), the matches are displayed as different bars in the same line; that is, one line per sequence. Selecting any hit in the Map View selects the corresponding row in the Table View.

Note. The order of the hits in the Map View is the same as in Table View, with initially the best hits colored yellow and appearing at the top of the view.

Return to the Table View and click any column header.

This sorts the hits in the table according to the selected column. When the Table View is sorted, the order of the hits in the Map View is changed accordingly.

Switch back to the Map View and verify that the order of the hits has changed to mirror the order in the Table View.

Choose View | Zoom | In from the menu bar or press CTRL + + on the numeric keypad to zoom in on the Map View.

This allows you to see the residue display on the ruler at the top of the window. You may have to zoom in several times to see the residue display.

4. Locating and opening the selected structure

Because one of the goals of this lesson is to create a 3D model of the protein sequence virtual-pharma-seq, a suitable homolog or template must be found. An ideal homolog covers the entire sequence length, has a fairly high sequence identity, and has a good E-value (< 1 x 10-5). This reduces the number of qualifying candidates to half of the hits obtained from the BLAST run.

Note. When an actual crystal structure of this protein sequence has been solved, this lesson will change. Watch for sequence identity scores greater than 95%.

Another goal of this lesson is to determine the active site (or interface) residues of the protein with a bound inhibitor, so a PDB structure that was crystallized as a complex with an inhibitor must be selected, such as 1B2Y or 1BVN. It is clear from most of the entries which have low E-values that the protein is similar to α-amylase, so this function can be inferred on virtual-pharma-seq.

The 1b2y structure is not ideal because this amylase protein was solved in the presence of a carbohydrate inhibitor (less interesting from a drug development perspective). The 1BVN structure is more suitable. This amylase protein is crystallized with a proteinaceous inhibitor (Tendamistat). Thus the X-ray crystal structure of 1BVN will be used as a template (homolog) for the modeling protocol and to characterize the interface between the amylase protein and its proteinaceous inhibitor.

Click the header of the Accession column in the Table View to sort the table by name.

Choose Edit | Preferences... from the menu bar to display the Preferences dialog. Choose 3D Window | Import from the tree view on the left side of the dialog to display the Import sub page. Check the Use PDB Secondary Structure checkbox and select the Kabsch & Sander option. Click OK to close the Preferences dialog.

The Use PDB Secondary Structure option instructs Discovery Studio to read the secondary structure assignments defined in imported .pdb files.

Before loading the PDB structure of the template, the location of the PDB file should be specified using preference settings. If you have WEB access, you can retrieve PDB file from a PDB hosted web server. Otherwise, you can point to the tutorial data directory that you downloaded.

Choose Edit | Preferences to display the >Preferences dialog. Expand the Files Explorer and select PDB Location. To use the web site, select the Web Site option and select pdb.rcsb.org from the drop down list. To use the local PDB file, select the Local File Path option and enter the full path in the text box. Set the rest of the parameters as follows: In Sub-Folder unchecked; Prefix set to none; Suffix set to pdb; and Compression set to none.

In the Table View of the BLAST Results Window, click the hit 1BVN_P to select it. Right-click the selection and choose Load Selected Structures from the context menu.

Note. The location of the PDB database may need to be set. To set the local file path, go to Edit | Preference | Files Explorer | PDB Location.

This opens the complete structure of 1BVN in a 3D Window.

In the virtual-pharma-sequence Sequence Window, right-click and choose Insert Sequence | From Windows... from the context menu to display the Insert Sequences From Windows dialog. Select 1BVN from the list and click the OK button.

This adds the 1BVN structure, with appropriate secondary structure information, to the Sequence Window.

You can now close the virtual-pharma-seq Blast Window. Select File | Close when this window is active.

5. Aligning the sequences using the Multiple Sequence Alignment protocol

This section will demonstrate how the model sequence can be aligned with the template sequence using the Multiple Sequence Alignment protocol. This step is necessary before building a model from the virtual-pharma-sequence based on the 1BVN template.

In the virtual-pharma-sequence Sequence Window, the Sequence Identity, ~6%, and the Sequence Similarity, ~18%, are shown in the Status Bar (in the lower right corner of Discovery Studio). Both values will increase after the alignment step.

The residue background is colored according to the residue similarity. Note the distribution of colors. You will see that more residues have a background color of dark turquoise (indicating identical residues) after the alignment step.

Note. If the coloring does not automatically appear, right-click in the Sequence Window and chose Display Style (or CTRL + D in the Sequence Window). Select the Background Color tab and click Color By and select Sequence Similarity. Note that you can change the coloring scheme here by clicking the colors in the Color column. Click OK.

Tip. Monitor the sequence identity/similarity scores and color pattern of the residues in the Sequence Window. If you did not wish to use the Multiple Sequence Alignment algorithm, you could align the sequences manually by inserting gaps. Click in the Sequence Window at the place you wish to insert a gap and then press the SPACE bar.

In the Protocols Explorer, double-click the Multiple Sequence Alignment protocol under the Protein Modeling protocol group.

In the Parameters Explorer, click the cell in the Parameter Value column for the Mode parameter and select Align Sequences from the dropdown list (this should be the default value). Set the Parameter Value for the Sequence Window 1 to virtual-pharma-sequence and Sequences in Sequence Window 1 to 1BVN and virtual-pharma-seq,respectively. They should automatically be checked.

The required parameters (those initially highlighted in red) are now set.

Click the Run button on the Parameters Explorer.

After the Multiple Sequence Alignment protocol finishes (it should take about 10 seconds), a new Sequence Window, titled virtual-pharma-sequence(1), is displayed, showing the aligned sequences.

The Sequence Identity (~37%) and Sequence Similarity (~48%) scores are now higher. You should also note that there are now more residues colored dark turquoise, indicating strong similarity in those regions.

Right-click in the Sequence Window and choose Secondary Structure Cartoon from the context menu.

This allows you to view the PDB and Kabsch-Sander secondary structure of 1BVN under the 1BVN sequence in the Sequence Window.

Note. You may need to enable the display of PDB and Kabsch-Sander secondary structure:

1. With the Sequence Window virtual-pharma-sequence(1) active, choose View | Display Style... from the menu bar to display the Display Style dialog.

2. Select the Display tab and check the PDB and Kabsch-Sander checkboxes. 3. Click OK.

The two types of secondary structure differ slightly. Observe that most of the gaps are inserted in the loop region of 1BVN.

In the Jobs Explorer, select the Multiple Sequence Alignment job and click the Locate button.

This locates the output folder containing the automatically saved input and output data for the protocol in the Files Explorer. We do not need to open any file from this folder at this stage of the lesson, but if you need to locate the experiment folder, this is the easy way to do it as mentioned before.

6. Manually adjusting the alignment

Expand the Hierarchy View in the 3D Window containing the 1BVN structure, then switch to the virtual-pharma-sequence(1) Sequence Window.

Note. if the Hierarchy View is not visible, select View | Hierarchy View and this view should appear on the left side in the 3D Window.

You should be able to see from the Hierarchy View that there are two protein chains - the first P chain for the α-amylase and the T chain for the inhibitor. Notice that the first P chain and T chain are separated by a chain break, shown as a red backslash symbol in the Sequence Window. Additionally, two ions are ligated by the P chain, Ca2+ (represented as another P chain in the Hierarchy View) and Cl- (represented as <Chain> in the Hierarchy View).

View the chain id of the Val804 residue immediately after the chain break by selecting and then hovering the mouse over that residue.

The popup shows T:VAL804, giving the chain ID, residue name, and its position. Clicking the residue updates the Status Bar with the same data.

A few residues at the C-terminal end of virtual-pharma-seq are aligned to the inhibitor, the T chain, of 1BVN. This indicates the alignment in this region is not accurate and needs manual adjustment.

DKVGNSCTGIKVYVSSDGTAQFSISNSAQDPFIAIHAESKL/VSEPA

------------------PASTATSWGAMTTAAAVMSPSTA-AARPA

Select the highlighted gap positions in the virtual-pharma-sequence(1) Sequence Window as shown in the above alignment by dragging the left mouse over the gaps, then right-click and select Gaps | Remove from the context menu so that the alignment at the C-terminal end becomes:

NSCTGIKVYVSSDGTAQFSISNSAQDPF

PASTATSWGAMTTAAAVMSPSTAAARPA

Note you can also click Delete when the gaps are selected. Also, if you made a mistake you can use the Undo command.

The inhibitor is now not aligned with the virtual-pharma-sequence and the Sequence Identity (~38%) and Sequence Similarity (~49%) scores are slightly higher when compared to the automatic sequence alignment.

7. Building a 3D model

In this section, you will build a 3D model of your original protein sequence using the alignment you just created and the 1BVN structure as the template.

In the Protocols Explorer, double-click the Build Models protocol under the Protein Modeling protocol group.

In the Parameters Explorer, set the Parameter Value for the 3 required parameters - Alignment, Model Sequence, and Protein Structures - to virtual-pharma-sequence(1), virtual-pharma-seq, and 1BVN, respectively.

Set Optimization Level for Model parameter to Low.

This allows the job to run faster.

In the Protocols Explorer, click the Show All button in the top left corner.

This displays all of the parameters for the Build Models protocol.

In the Parameters Explorer, set the Parameter Value for the Cut Overhangs parameter to True.

Note. Select another parameter to make sure this is set to True before proceeding.

This removes any terminal unaligned residues from the model sequence.

Click the Run button on the Parameters Explorer.

During the calculation, the Jobs Explorer monitors the status of the job. This should take about 3 minutes. You can continue to the next section while waiting for the job to complete.

8. Determining the binding interface residues

In this section, you will identify the residues on the α-amylase/inhibitor interface of 1BVN. You will then create two groups of residues:

• A group of the few key interacting residues from the inhibitor (T chain) • A group of the P chain of the α-amylase protein

The interface is the most interesting region for designing a small molecule inhibitor. After running this protocol, you should compare the 1BVN structure to your model, focusing on the interacting residues to make a direct comparison of these residues in type and 3D location.

In the 3D Structure View for 1BVN, right-click and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog.

Click the Atom tab and select the None option from the Display Style control group.

Click the Protein tab and select the Solid Ribbon option from the Display Style control group. Click OK.

This turns off the display of individual atoms and represents the protein backbone as a solid 3D ribbon.

In the Hierarchy View, click Water and press DELETE.

All water molecules in the structure are deleted.

In the Hierarchy View, identify the inhibitor, the T chain, and expand it to reveal the T-chain residues. Select the following residues by pressing and holding CTRL while clicking: TRP818, ARG819, TRY820, and TYR860.

These residues are highlighted in the virtual-pharma-sequence(1) Sequence Window and the 1BVN 3D Structure View and Hierarchy View.

In the 3D Structure View, right-click and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog. Click the Atom tab and select the Stick option from the Display Style control group. Click the OK button.

The selected atoms are displayed as solid cylinders.

With the atoms still selected, choose Edit | Group... from the menu bar to display the Edit Group dialog. Enter the name epitope in the Group Name text box and click the Define button.

This action creates a group called epitope from the four selected residues that is now visible in the Hierarchy View.

Select the epitope group in the Hierarchy View and choose Edit | Select... from the menu bar to display the Select dialog. Check the Radius checkbox and enter a value of 6 in the associated text box and check the Type checkbox and select AminoAcid from the dropdown list. Click the Select button.

This selects all the amino acids within 6 Å of the epitope residues.

Within the Select dialog, check the Type checkbox and select AminoAcidChain from the associated dropdown list. Check the Name checkbox and enter T in the associated text box. Click the Deselect button.

This removes the selected atoms from the T chain (inhibitor) and leaves only the P chain atoms within 6 Å of the selected epitope residues. These are the atoms involved in direct binding and hydrogen bonding to the inhibitor epitope and are useful for designing a pharmacophore query.

Click the Close button on the Select dialog.

With the remaining epitope atoms still highlighted, select Edit | Group... from the menu bar to display the Edit Group dialog. Enter bzone in the Group Name text box and click the Define button.

In the 1BVN 3D Structure View, right-click and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog. Click the Atom tab, and select the Stick option from the Display Style control group. Click the OK button.

Now you will enable hydrogen bond monitors.

Click epitope in the Hierarchy View to select the residues in this group. Choose Structure | Monitor | Intermolecular HBonds from the menu bar. In the Hierarchy View, select HBond Monitor1 and click the

Fit To Screen button to zoom in on the monitors.

Note the hydrogen bonds, represented by dotted green lines (when not selected), extending from the inhibitor residues and the residues in the amylase protein. These are important query design elements.

Choose File | Save As... from the menu bar to display the Save As dialog. Select Viewer Files (*.msv) from the Files of type dropdown list and enter 1BVN.msv as the File name. Select a location on your machine for the file and click the Save button.

This saves the 1BVN structure with the groups and hydrogen bond monitors that you created as an .msv file

for later use.

9. Evaluating the model

In this section, you will compare the model structure with the template and evaluate the model score generated by the MODELER program.

Before continuing to the next step, you can close a few windows that are no longer needed:

• virtual-pharma-sequence Sequence Window • virtual-pharma-sequence(1) Sequence Window • virtual-pharma-seq BLAST Window

Click the Close button in the top right corner to close a window. When prompted to save the files, click the No button.

In the Jobs Explorer, right-click the Build Models job you set up and ran in step 7 and select Locate from the context menu to open the output folder for the protocol in the Files Explorer.

With the 1BVN 3D Structure View active, choose File | Insert from | File... from the menu bar to display the Open dialog. Browse to the Build Models output folder and select the virtual-pharma-seq.B99990001.msv file. Click the Open button.

The model structure is added into the 1BVN 3D Window.

In the Files Explorer, double-click the virtual-pharma-sequence.bsml in the output folder within the Build Models experiment folder.

This opens the alignment file of the model and the template sequences. Three sequences are displayed as aligned in the Sequence Window, two of the query sequences, virtual-pharma-seq and virtual-pharma-seq.B99990001, and one of the template, 1BVN.

Note. The sequences, 1BVN and virtual-pharma-seq.B99990001 are automatically linked to the corresponding structures in the 3D Window.

Click 1BVN in the Hierarchy View to select it and choose Structure | Superimpose | Superimpose by Sequence Alignment... from the menu bar to display the Superimpose by Alignment dialog. Select virtual-pharma-sequence as the Sequence Alignment and virtual-pharma-seq.B99990001 as the Molecule to Superimpose. Click the OK button.

The two structures are superimposed and a text report is generated automatically. If you check the report, you should find that the RMSD between the alpha-carbon atoms of the two proteins is ~2 Å over 424 aligned residues. Close this window.

With nothing selected in the 3D Structure View, right-click and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog.

Click the Atom tab and select the None option from the Display Style control group.

Click the Protein tab and select the Solid Ribbon option from the Display Style control group. In the Coloring control group, select the Color By option and choose CAlpha from the dropdown list. Click the OK button.

You should be able to see clearly that the two proteins are superimposed on top of each other.

Choose View | Tile Molecules in View from the menu bar.

The two structures are shown side by side in the 3D Structure View. You can rotate and move them in sync with the normal rotate and move mouse actions.

With the 3D Structure View active, choose View | Data Table from the menu bar.

This displays the Data Table View and allows you to further evaluate the model.

With the Data Table View active, click the AminoAcid tab. Click in the PDF Total column header to select it and choose Chart | Simple Line Plot from the menu bar.

Two line plots are displayed. The 1bvn-Line Plot window shows an empty line because the template structure does not have any PDF energy. This window can be closed. The 1bvn-Line Plot(1) window shows the per-residue total PDF energy against the residue index for the model structure. You should see a big peak at a PDF total score of close to 200.

Click and drag around the tall peak at about 200 to select it.

The selection consists of the residues with highest scores. The selected residues are highlighted in the 3D Structure View and in the Sequence Window. The high PDF energy indicates bad alignment for those residues. The alignment needs to be manually adjusted, so the model needs to be rebuilt, but you will not do this in this lesson.

In the Hierarchy View, select the bzone group on the template structure.

The binding site residues are highly conserved and aligned well in the sequence alignment. The PDF energy for those aligned residues is quite low, which indicates good structural agreement around the binding site.

Close all the Sequence and Structure Windows. When prompted to save the files, click the No button.

10. Assessing the validity of the 3D structure using the Verify Protein protocol

The model structure can be further evaluated using the Verify Protein protocol, which assesses the compatibility of the 3D structure of a protein model with the sequence of residues it contains.

In the Jobs Explorer, double-click the Build Models job you set up and ran in step 7 to open the output folder for the protocol in the Files Explorer. Double-click the virtual-pharma-seq.B99990001.msv file in the Files Explorer to display the model structure in a 3D Window.

In the Protocols Explorer, double-click the Verify Protein protocol under the Analysis protocol group.

In the Parameters Explorer, click the cell in the Parameter Value column for the Protein Structure parameter and select virtual-pharma-seq.B99990001:virtual-pharma-seq:B99990001 from the

dropdown list. Click the Run button.

The calculation should finish in ̃ 15 seconds. When it is complete, a new 3D Window is opened containing the model structure. The structure is displayed as a solid ribbon with variable width and color based on the Verify score; the higher the score, the better the structure. The color varies from blue to white to red, with blue corresponding to high scores, white to average, and red to low scores. The width of the ribbon is anti-correlated with the Verify score; the worse the structure, the wider the ribbon.

In the Data Table View, click the Molecule tab and scroll to the end of the table to the Verify Score, Verify Expected High Score, and Verify Expected Low Score columns.

If the Verify Score result of the model protein is higher than the Verify Expected Low Score value, then this indicates that the model is of acceptable quality. The closer the Verify Score result is to the Verify Expected High Score value, the better the quality of the model, which in our case is fairly good.

In the Data Table View, click the AminoAcid tab and scroll to end of the table to the Verify Score column for the residues. Click the Verify Score column header to select it and choose Chart | Simple Line Plot from the menu bar.

The plot shows the score for each residue in sequence. Note the low score region towards the C-terminal end of the protein.

Open the virtual-pharma-sequence.bsml file in the output folder from the Build Models job. Click and drag around the low score peak to select the residues with the lowest scores in the line plot.

The selected residues are highlighted in the 3D Structure View and the Sequence Window, allowing you to see the low scoring region on the alignment.

The selected region with the low Verify Score values is in the vicinity of the residues which had high PDF energies. This indicates that the alignment in this region may need further adjustments and that the model should be rebuilt.

The next step is optional.

11. Calculating electrostatic potential using DelPhi (optional)

In this section, you will set up and run the DelPhi Electrostatic Potential protocol to calculate an electrostatic map of your protein model. You will also analyze the surface properties of the protein in the context of charged surface residues. Different surface visualizations will be used to help analyze the electrostatic properties of the protein.

Close any open windows remaining from previous sections. Open the model file, virtual-pharma-seq.B99990001.msv, in the output folder from the Build Models job.

In the Tools Explorer, double-click the Electrostatics tool panel to expand it. With the 3D Window active, select the Parse command from the Apply Templates tool group.

Note. The Tools Explorer window can be opened by selecting View | Explorers | Tools. The Electrostatics tool panel can be displayed by selecting View | Tool panels | Electrostatics.

This assigns the Parse atomic charges and radii template to the protein model.

Note. Hydrogen atoms are automatically added to the protein structure, where appropriate, when the charge template is applied.

Select the Atom tab of the Data Table View. Scroll to end of the table to the DelPhi Radius and DelPhi Charge columns.

Note that DelPhi radii and charges have been assigned to each atom in the protein.

Select the List Net Charge and Residue Charges tool from the General Tools tool group on the Electrostatics tool panel.

A text report listing the net charge of the protein and of each residue is produced. You should find that the net charge on every residue is an integer value.

In the Protocols Explorer, double-click the Electrostatic Potential protocol under the Analysis protocol group.

In the Parameters Explorer, click the cell in the Parameter Value column for the Input Model parameter and select virtual-pharma-seq.B99990001:virtual-pharma-seq:B99990001 from the dropdown list.

Leave the remaining parameters set to their default values. Click the Run button.

This starts the Electrostatic Potential calculation, which will take ˜1 minute to complete.

When the job is completed, select the Electrostatic Potential job in the Jobs Explorer and click the Locate

button to open the output folder for the protocol in the Files Explorer. Double-click the virtual-pharma-seq.B99990001.msv file in the Files Explorer to display the model structure in a 3D Window.

The protein structure is displayed with an isosurface of isovalue of -1.0.

In the Hierarchy View, select the isosurface virtual-pharma-seq.B99990001.

The isosurface is highlighted in the 3D Structure View.

In the 3D Structure View, right-click and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog.

Click the Isosurface tab and select the Custom option from the Coloring control group. Click the Color chooser and select red from the color palette that is displayed. Click the OK button to close the color palette. Click the OK button on the 3D Structure View Display Style dialog.

The isosurface is colored red.

With the isosurface selected, choose Structure | Isosurface | New Contour... from the menu bar to display the Add Isosurface Contour Level dialog. Enter a value of 1.00 in the Isovalue text box and click the OK button.

A new isosurface is added to the structure.

In the Hierarchy View, select the new isosurface, then right-click in the 3D Structure View and choose Display Style... from the context menu to display the 3D Structure View Display Style dialog.

Click the Isosurface tab and select the Custom option from the Coloring control group. Click the Color chooser and select blue from the color palette that is displayed. Click the OK button to close the color palette. Click OK on the 3D Structure View Display Style dialog.

The new isosurface changes color to blue. Now the positively charged portion of the protein is covered by the blue isosurface and the negatively charged portion by the red isosurface.

You can also color a molecular surface using the DelPhi grid value.

Choose Structure | Surface | Add... from the menu bar.

This displays the Create Surface dialog.

On the dialog, select Solvent surface and click the OK button.

This adds a molecular surface to the protein.

In the 3D Structure View, right-click and choose Display Style... from the context menu.

This displays the 3D Structure View Display Style dialog.

Click the Isosurface tab and select the Off option from the Display Style control group to turn off the isosurface display.

Click the Surface tab and select the Solvent option from the Display Style control group. Select By Grid from the Coloring control group and click the Colors button under that to bring up the Color Mapping dialog box.

In the Color Mapping dialog box, select Red-White-Blue from the Spectrum dropdown list and enter 16 in the Bands parameter. Right-click and drag the two vertical lines (which may appear as one in the center of the peak) in the Histogram window to focus on the peak value in the histogram. Click OK button to close the dialog box and then click Apply button in the Display Style dialog box.

The solvent surface is displayed using the color spectrum that you specified. You may turn on the transparent option to see different parts of the structures under the surface.

The Electrostatics analysis can also be done on the template and compared to the model electrostatics result you just performed.

This is the end of the lesson.

Discovery Studio Tutorials - ndmctsgh.edu.t 1: Working with the mouse, the Sequence Window, and the...

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