1. Open the SPDBV_4.04_OSX folder on the desktop and double click DeepView to open.
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1 Molecular of inhibitor-bound Lysozyme This lab will not require a lab report. Rather each student will follow this tutorial, answer the italicized questions (worth 2 points each) directly on this protocol/worksheet, and turn this in at the end of the lab period. In addition, have the top of this page initialed by your instructor to confirm that you have successfully checked out of lab (1 point). Check out complete? 1. Open the SPDBV_4.04_OSX folder on the desktop and double click DeepView to open. 2. Cancel the dialog box that automatically opens. 3. Click File: Import and type 1HEW in the name box. Click PDB file under the grab from server portion on the webpage. The model, in stick form, should appear. You should be looking at a red, blue, white and yellow stick model on a black background. Try to locate the inhibitor, which is composed of three chair-form rings of N-acetylglucose. It's not easy. Coming sections of the tutorial will show you how to highlight or single out specific features of a model and make them jump right out at you. 4. If a window named inputlog.txt is open, close it. In order to streamline the initial program operation, change a few settings: Uncheck "Show the 'setting depth to 256 colors'..." Uncheck "Show splash screen at startup." Uncheck "Show log file upon loading." Click OK. On startup, DeepView opens two windows, the display or graphics window, which is labeled with the name of the PDB file (and the window size in pixels), and the Control Panel (on the right if the Control Panel does not appear, use the command Wind: Control Panel to display it). The Control Panel lists the amino-acids residues and other contents of the PDB file. You use the Control Panel to select residues, establish the content of the display, label residues, and color them. Above the graphics window is the Tool Bar. You use the graphics window and the buttons on the Tool Bar to view, manipulate, and measure the model. You must click on a window to make it active. 5. Display additional windows: Wind: Sequences Alignment The narrow Align window appears below the graphics window, showing the amino-acid sequence of the protein in one-letter abbreviations. You use this window when comparing sequences of two or more proteins. Select: All The color of residues listed in the Control Panel changes from black to red (and in the Sequences Alignment window, from white to purple). The new color means that a residue is currently selected. Some DeepView commands affect only the selected residues. 1
2 Wind: Ramachandran Plot The Ramachandran Plot window appears in the upper left. You can use this window to judge the quality of a model, by finding residues whose conformational angles lie outside allowed ranges. You can also display and change conformational angles in the model. Each dot (actually, a tiny cross or square) on the diagram gives the phi and psi angles of one residue in the protein. Because all residues are selected, all are represented in the window. Wind: Layer Infos The small Layer Infos table appears at the upper right. This table provides control of multiple protein models, allowing you to choose which models are visible, which models can move, and which models have certain features on display. 6. On the Tool Bar, below the first square button on the left (Zoom and Center), are two symbols, a globe and a little dog-eared piece of paper (document icon). Click the document icon to see the PDB file of the protein currently on display. This file will come to mean more to you late. Close the file window before proceeding. Close the Align, Ramachandran Plot, and Layer Infos windows. 7. Center the model by clicking the first button on the left of the tool bar, Zoom and Center (or press = on the keyboard). Control the movement of the model by clicking through the next three buttons. 8. Turn on stereo display by clicking Prefs: Stereo Display. The Allow Real Time Stereo Display box should be checked and Use Side by Side Stereo should be selected. Set the Rotation angle to for cross-eyed viewing and set the Stereo Separation to 180 pixels. Adjust if you need more or less space between the pictures. Click OK. Switch between stereo display and normal display by clicking on command (apple key), t. 9. Click anywhere on the Control Panel window to make it active. First, simply scroll down to the bottom of the list to see how many amino-acid residues the protein contains and to see how the hetero groups are named at the bottom of the list (hetero groups are always listed last). * How many amino acids are present in the model? 129 *The tri-nag inhibitor are three sequential residues in NAG. What are they numbered as? Now click and drag, starting on the GLY4 near the top of the window and finishing at GLY16 (this will include amino acids at position 4 16). All group names from GLY4 GLY16 should turn red and are now selected. Press return on the keyboard. Zoom and center. *What type of secondary structure are you viewing? Alpha-helix 2
3 11. Notice also that a checkmark has appeared in the show column next to the selected residues, indicating that they are on display. There are checks also in the side column, which means that side chains are shown. A side chain is shown only if the rest of the residue is shown, so you only see the side chains of displayed residues in the graphics window. 12. Click the check in the side column next to MET12, to hide the side chain. Click the same space again to replace the check and display the MET12 sidechain. Try the same thing in the show, label, surface (little circle of dots), and ribn columns. Surface displays a dotted van der Waals surface for the group. Ribn (ribbon) draws a smooth-stranded cartoon of the selected backbone. *What element does the yellow color represent? Sulphur *Is there a second residue shown that contains this same element? Yes *If so, which residue is it (indicate the name and location)? Cys6 13. Remove the labels, surface, and ribbon as follows: hold down the shift key and click any checkmark in the label column. Repeat for the surface and ribn columns. When you click on a checkmark while holding down shift, your action removes all checkmarks, and DeepView makes the corresponding changes in the display. When you shift-click an empty space, DeepView places checkmarks in that column beside all groups and takes the approprate action. Shift-click several times in the show column. This displays and hides the whole model. With all groups shown, shift-click several times in the side column to display and hide side chains. End with side chains hidden. Shift-click in the ribn column to display and hide a ribbon cartoon for the whole model. Remember that you can center the display at any time with Zoom and center. 14. Select and show residues 4-16 with their side chains, and add residues 17 through 22 as follows: click in the show column next to LEU17, drag down the column to GLY22, and release the mouse button. Arrows should appear in the show column next to residues 17 through 22, but these residue names should not turn red. Add checkmarks in the side column for residues in the same way. Now residues 4 through 16 are selected and shown, while residues 17 through 22 are shown, but not selected, press enter on the keyboard. *What type of secondary structure do you observe in the added residues? Beta turn/coiled coil not an alpha helix or a beta sheet 15. Notice the result on both the Control Panel and the model. Press enter again. The enter key toggles the display of selected groups on and off. Press enter several times, and be sure you understand what is happening. End with residues 4-22 displayed, but only residues 4-16 selected. Now press return. This shows all selected groups, and hides all unselected groups. 3
4 Notice the difference between the return and enter keys; the return key adds selected groups, and removes unselected groups, while the enter key toggles between showing and hiding selected groups, without affecting unselected groups. The + and - symbols above the Control Panel column headings act as buttons to turn the column function on or off for the selected groups. 16. You can also select groups of residues that are not consecutive. With residues 4-16 selected and shown, hold down the ctrl and click TYR20 and LEU25. This adds residues 20 and 25 to the selection (shown in red). Press return to add them to the display. These residues are disconnected from the others. If you do not see them, Zoom and center. Click the word side at the top of the Control Panel. This adds side chains to the newly selected groups. Note again that clicking headings in the Control Panel affects the selected residues only. 17. There are also two narrow columns to the left of the group column. The first column is blank when the current model contains only one protein chain, as does 1HEW.pdb (if the model contained more than one chain, the one-letter chain designations would appear in this column). Click anywhere in the first, blank column. You have selected the entire chain (in this case, the entire model). Press return and then help or = to see the entire model. 18. Find and display all potential hydrogen bonds in the molecule, including those involving side chains and hetero groups with Tools: Compute H Bonds. DeepView displays hydrogen bonds as green dashed lines. 19. To see only the alpha-helical regions of lysozyme with hydrogen bonds shown, choose Select: Secondary Structure: Helices return. Turn off all side chains. Look around to confirm that the segments shown are helical. Confirm that most of the hydrogen bonds are from the carbonyl oxygen of residue n to the amide nitrogen of residue n + 4. Notice the Control Panel: only the residues in helices are red. This allows you to take further action on the selected groups, such as coloring them, as you will see in the next section. *How many alpha-helices are present? To see only strands of beta pleated sheet, choose Select: Seconday Structure: Strands return. Notice the characteristic pattern of hydrogen bonds in the strands. *Which strands are parallel, and which are antiparallel, to each other? LYS1 through PHE3 are antiparallel to PHE38 through THR40 ALA42 throughasn46 antiparallel to SER50 through GLY54 which are, in turn antiparallel to GLN57 through SER Display: Show H Bonds You will find that this command has a checkmark to its left, which means it is turned on. By selecting it, you turn it off, and remove the hydrogen bonds from the display. 22. Notice the other Select menu commands. Can you select and display only the residues whose side chains carry positive charge (so-called basic residues)? Can you select and display only the histidine residues and their side chains? (Look at the submenu that pops up when you choose Select: Group Kind.) 4
5 *How many histidines are present in lysozyme? The Layer Infos window provides information about the display. Select Wind: Layer Infos. Note the Sel heading at the top of the far right column. This column shows the number of groups currently selected. Again select the basic residues, and notice the number selected under the Sel heading. Now select acidic residues. Compare the number of acidic and basic residues in lysozyme. Put away the Layer Infos window by clicking on its close box, the small square in its upper left corner. * Would you expect the isoelectric ph or pi of lysozyme to be above or below ph 7? It's around ph 10, because basic side chains outnumber acidic ones. 24. Select only groups NAG and then press return and help. You should see tri-nag only. Manipulate and get to know this trisaccharide structure. 25. Another powerful selection tool for Atom selection lies at the top of the graphics window. It is the ninth button from the left, showing an eye and a circle. Click this button. In the message space just beneath the movement icons, DeepView instructs you to pick an atom. Click the nitrogen atom (blue) on the center ring (NAG 202). A dialog appears: Click to darken the first button (add to display), type the number 8 in the box and click OK. You have added to the display all groups having at least one atom within 8.0 angstroms of the atom you picked. (You may need to click the side heading in the Control Panel to add the side chains.) Other buttons in the dialog allow you to select the neighbors or to display only the neighbors. In addition, Select:Visible Groups allows you to change the current selection to include all groups on the screen. 26. To display all groups within a specified distance of the selected groups, select and display only NAG Then, Select: Neighbors of Selected aa... In the dialog, type the number 4.5 in the box and click OK. You have added to the display all groups having at least one atom within 4.5 angstroms of any atom in NAG Display: Show H-Bonds (you computed H-bonds earlier, and DeepView still remembers them). 27. Now select NAG (select only -- do not remove other groups from the display). Display: Show Only H-Bonds From Selection then Display: Show Only Groups With Visible H-Bond help. With this simple set of commands, you have limited the display to the tri-nag inhibitor and residues to which it is hydrogen bonded. 28. Select: Visible Groups then Select: Inverse Selection with the heading: ribn press help. Now you should have a model of lysozyme in which tri-nag and its hydrogen-bonded groups are shown in wireframe, with all the rest of the model shown as a ribbon. *Which side chains are interacting with NAG? ASN59, TRP62, TRP63, ASP101, ASN103, ALA107 5
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