Discovery Studio 1.5. Online tutorial. Standalone server documentation

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1 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 Lesson 1 - Working with the mouse, the Sequence Window, and the 3D Window Lesson 2 - Working with structural models Description Provides an introduction to mouse commands, display styles, their interactions with the Sequence and 3D Windows, and to aligning multiple protein structures. 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 Lesson 5 - Fitting single residues Lesson 6 - Homology modeling of an extracellular amylase protein Standalone server documentation 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. 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. 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. 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

2 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 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.

3 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

4 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.

5 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

6 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.

7 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.

8 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. Modules: Discovery Studio Visualizer 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 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.

9 2. Open an insulin crystal structure Choose File Open... from the menu bar. This displays the Open dialog. On the Open dialog, navigate to and select the 9INS.pdb file. Note. Instructions for obtaining data files necessary to running this and other tutorials are available at 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

10 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 C α 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 C α 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 C α 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 C α 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.

11 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 C α 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 C α atom of CYS(A6). 2. Create a 3D text label near a disulfide bridge In the 3D Structure View, click the γ-sulphur atom, S γ, 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 C α atom of ASN(A21). Click the Add Label... command in the 3D Labels tool group to display the Add Text at 3D Pointer dialog.

12 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 Click the Open button.

13 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.

14 An NCS matrix named B_to_D_Ins and based on the equivalent segments that you specified is generated and stored. The C α 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 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 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

15 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 C α 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 C α representation of crystal packing

16 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 C α representation of the crystal packing of the insulin molecules within a unit cell. Each molecule is represented by a different color, as before. This is the end of lesson 2. Lesson 3: Building and editing small molecules Purpose: Introduces the sketching tools. Modules: Discovery Studio Visualizer 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

17 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 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. 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.

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

19 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.

20 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. This is the end of lesson 3. 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

21 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. Choose File Open... from the menu bar. This displays the Open dialog. Note. Instructions for obtaining data files necessary to running this and other tutorials are available at 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.