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Molecular Visualization using Swiss PDB Viewer OUTLINE Step 1: Searching the PDB to obtain PDB files Step

2: Loading a PDB file into Deep View Step 3: Working with Deep View Tools The Control Panel The Main Toolbar: Rotation, Zooming and Centering Changing colors Exercise 1 Step 4: More Viewing Tools: Slabs, Selecting Specific Residues and Ramachandran Plots

Step 1: Searching the Protein Data Bank to obtain PDB files
To start, we need a PDB file and the Swiss PDB-Viewer. Open a web browser of your choice and follow the link below to the Protein Data Bank website. Also open the Swiss PDB Viewer program. 1. Go to the Protein Data Bank.
This is THE repository for protein structural information. Crystallographers and other structural biologists enter the data they have collected on their protein structure in this database. Each entry in the database is given a 4 character alphanumberic identification number. This number is unique for each structure and is used to file it in the database. Associated with each entry in the database is a series of information relating to that protein.

2.

Find the PDB entry for bovine beta-trypsin.

You may do this in two ways (try both): 1. In the search box enter bovine beta trypsin and press the search button. You should find that there are many entries for this query. These include many structures of bovine betatrypsin, trypsin inhibitors and also structures with bovine beta-trypsin complexed with several inhibitors. We want a particular structure so follow step 2.

2. In the search box enter 1TPP

You should find one entry on the results page. This is the entry that we will work with during this exercise. You will need to download the .pdb file, get information about this structure from the .pdb file and view the structure using the Swiss PDB Viewer. 4.

Step 2: Loading a PDB file into Deep View 1. Open the Swiss PDB-Viewer. If you do not have this program on your computer you can download it from the SwissPDBViewer site. It is available for Mac and PC. The controls for these programs look similar, but are not entirely identical. This tutorial was generated using the Mac version that we have loaded into the computers in the CCMML. 2. Run the SwissPDB Viewer program (choose the appropriate folder in the Applications folder on the Mac, select the appropriate folder on a PC). You should see a program bar appear that looks like this:

3. In the file menu click on Open in the menu options at the top of the screen and choose the 1TPP file you just downloaded. 4. The structure of 1TPP should appear as well as a text file (a log file) that will appear on top of the program bar. Close this text window so that what you see is the program bar and the stickmodel of the protein structure in the window below the bar. You should now have something that looks like this:

5. To view the .pdb text file for this structure click on the little "page" icon in the top left of the

program bar. This opens the .pdb text file in a new window. You can get a lot of information about your protein and this structure from this file. You will need a lot of this information to complete the protein structure assignment. On the page with the pdb file listed, you can explore the raw data that will be converted into your great-looking 3-D images in a couple minutes. This may look confusing at first. Remember that this info is set down in a particular format so that all structures can be displayed, archived and understood in the same fashion. Some of the important line items and their

descriptions are listed below: A listing of all the atoms in the structure, the amino acid or heteroatom that they belong to and the coordinate s of those atoms. Location of disulfide bonds Residues that are included in sheets Residues that are included in helices Heteroato ms are listed here. These are atoms that are not part of the primary sequence of the protein. These can include cofactors, metals or bound inhibitors. The primary sequence of amino Information given

acids in the structure This section contains additional informatio n about the structure. It includes all the informatio n the authors feel is necessary to properly cite, describe or explain the structural informatio n. Where this work was published The investigato rs that performed the study The organism the protein comes from The protein that is described Term ATOM SSBOND SHEET HELIX HET SEQRES REMARK

JRNL AUTHOR SOURCE COMPND Gives a basic description of the structure HEADER

Step 3. Working with Deep View Tools: The Control Panel, Rotation Tools and Changing Colors
Next we will work with this structure. Our initial goal will be the generation of a ribbon diagram and a space-filled diagram that highlights some key structural features of trypsin. It is helpful to be familiar with the basics of the mechanism of serine proteases. If you do not remember this from Chem 371 then review this in your Lehninger text. In this text the mechanism of the serine proteases is examined using chymotrypsin, the mechanism of trypsin is similar and also uses the classical catalytic triad (Ser, His, Asp) however the specificity of trypsin is different, it cleaves following basic amino acid residues. During our exercise we will examine the structure of the inhibitor that is bound within the active site and understand why it is bound where it is bound and what aspects of the natural substrate it mimics. NOTE: Depending on the speed of your computer and its graphics capabilities you may or may not want to toggle on the 3D rendering options in your window. To turn these features on go to the Display menu and turn on 'Render in Solid 3D' and 'Use OpenGL rendering.' You can play with these options to see which (or both on better computers) works best with your system. If neither works well, turning these features on only when you are finished working with the model (rotating, coloring etc.) or want to briefly see your model will work.

Deep View operates in as a series of windows. This is useful because you can open and close the windows as you need them. The main toolbar will always be open. It looks like this:

The other windows are opened from the Window menu ("Win") located at the top right. These will open as independent windows and can be opened and closed at any time. The Control Panel 1. Open the control panel

In the main toolbar, click on Window ("Win") and then Control Panel. The drop down menu is shown to the right. 2. The control panel, a long window will open to the right of the main window. The main features of this window are displayed in the aqua box below. Exercises to test your working knowledge and help in the completion of our initial tasks are indicated in the Exercise 1 yellow box below (be patient, there's a lot to get used to before you can work with the structure!). The Control Panel Explained The Explanation small letters to the left of the group name indicat e wheth er this residu e is locate d in a helix or a strand. These are differe nt from the capital letters that

can be locate d to the left of these letters that indicat ea separat e chain. Drop down menu for the color option. You use this drop down menu to choose wheth er you are changi ng the color of the backb one, backb one+si dechai n, sidech ain or the ribbon. 'col' stands for color. Highli

ghting particu lar residu es and clickin g on this box brings up a dialog ue box that allows you to change the color of the selecte d residu e. "ribn" stands for Ribbo n. If checke da ribbon will be traced throug h the backb one and it will be drawn as a helix or strand depen ding on the classifi

cation of that amino acid (see K) This symbo l stands for 'dots surfac e' or the van der Waals or accessi ble surfac e area of the protein . When checke d the vdW or accessi ble surfac e will be shown. The drop down arrow allows you to select which surfac e will be displa

yed. "labl" is short for Label. If this is checke d the amino acid will be labele d in the structu re with the three letter abbrev iation and the residu e numbe r. These labels can be change d in the Prefer ences menu "side" is short for side chain. If this is checke d then the

side chain for that particu lar atom will be shown. "show " is short for show backb one. If a "V" is listed (a check in Deep View) in this colum n then the backb one for that particu lar atom will be shown The 'group' colum n lists the amino acid (in order from n to C termin us)

with the hetero atoms at the bottom of the list. To the left of the group colum can also be a colum n with letters from A-Z. If there is more than one amino acid chain in a molec ule, this is shown by these letters. If checke d, you can move the structu re around Label K J

I H G F E D C B This check-box toggles the visibility of the structure in this layer A

This checks/unchecks ('V') next to a residue and activates the feature in that column This selects all groups that you click on option+click on a group while control is pressed. This is useful for selecting a group of residues and especially (control+click on a PC) useful if they are not continuous within the sequence. shift+click within a This checks/unchecks all residues in that column column within a layer This checks/unchecks all residues in that control+shift+click column within all layers (if working with within a column multiple layers) Control+Z Undo clicking within a row The Main Toolbar, Rotation, Zooming and Centering The main toolbar contains all the major tools for navigating the structure, moving the structure and changing your perspective. Label Description A Clicking on this button centers the molecule on the screen Clicking on this button changes your cursor to the 'hand' which allows you to move the B entire molecule or selection

C D E F

Clicking on this button changes your cursor to the zoom-in zoom-out cursor. Dragging your mouse forward and back will zoom you into and out of the structure. Clicking on this button changes your cursor to the rotate cursor. Moving your mouse side-toside and up and down will rotate the molecule or selection Clicking on this changes the rotation from a global rotation or a rotation about the axis of symmetry of the molecule. Often this is useful to play with when you are trying to get a particular angle and can't quite get the structure to move the way you would like. Toggles between moving the entire structure or just the selected residues.

Changing Colors You can change the color of individual residues, ribbons, loops and strucures by selecting them individually and in groups and then changing the color of the ribbon, sidechain, backbone etc. from the Control Panel. You do this by selecting the down arrow under the 'col' option to change the item you want to color and then use the small boxes next to the residue to change the color. There are other options, however that give you important information and are easier to work with than selecting groups within the control panel. These selections are found in the Color drop down menu in the Main Toolbar. The box below describes the most useful features of this menu. Label Information Colors the individual atoms A according to a standard color scheme Colors by the type of amino acid. Non-polar B = grey, polar =yellow, acidic = red, basic =blue. Colors by secondary structural element. Helices are red, sheets yellow C and loops white, by default. You may change these colors in the Preferences menu. D Colors by

secondary structure succession from N to C terminus. This colors by wavelength of the visible spectrum from N terminus = violet to C terminus =red.

Exercise 1: Using the above tools to view meaningful aspects of serine protease structure.
Click on Outline, Control Panel, Main Toolbar and Changing Colors to return to the appropriate sections above. Click on the Instruction icons above to return to this section.

Comments and Assignment Instructions (in bold) Remember to turn off the side chains using the control menu. To make the ribbons solid, go Prefs-->Ribbons and change the following options to make the ribbons solid:

1. Create a ribbon diagram of trypsin. Color it by secondary structure and then by secondary structure succession.

2. Place labels on the N-terminal and Check "labl" next to these residues in the Control Panel C-terminal residues

3. Make the heteroatom(s) Use checkmarks in the 'show' and 'side' columns for these heteroatoms. appear in ball and stick representation 4. Rotate the Save a copy of this view by selecting Save-->Image in the File menu. Name this file structure so that Initials01 (Example my first file was LML01). This can then be imported into the Protein you best see the Structure Exercise document. You will then write a descriptive caption for this picture inhibitor bound within the active that describes what is shown, highlighted and the overall purpose of the figure. site and zoom the structure in/out to Note: On a PC early versions of this program saved files as .tga files. These must be best view the active opened in a photo editing program and saved as a different filename. On a Mac, this site, while still doesn't happen. The files are saved as a .pict file. If you want to rename the file you can retaining the entire open it in Preview or other photo editing software and rename it as a .tiff, .jpg or .gif for protein in the import into a word processing program. frame. 5. Now show the structure of trypsin in space-fill Use the control panel to change how the structure is shown. showing the van der Waals surface. 6. Color the Remember to color both the backbone and side chain. residues by Type 7. Retain the inhibitor in balland-stick form so that it can be seen within the active site. Note the possible interactions Save a copy of this view by selecting Save-->Image in the File menu. You will write a between that caption for this as described in the Protein Structure Exercise document. inhibitor and the protein that can be seen through analysis of the information provided in this view.

Step 4: More Viewing Tools: Slabs, Selecting Specific Residue, Calculating H-bonds and Generating Ramachandran Plots
The goal of this portion of the tutorial is to get you used to some more advanced features in Deep View. This will allow you to select specific residues and allow you to display only those residues, or to color these differently or display them in a particular representation. You can also compute H-bonds to see which amino acids are connected non-covalently. If you combine these selection, image manipulation and computational tools with the skills you learned above you will be able to create images that convey a lot of structural information.

Ramachandran Plots In addition to providing a tool for viewing structures, Deep View also allows you to examine the phi/psi angles of the residues in the protein. In the Window menu is an option to generate a Ramachandran plot for the protein you are viewing. This is a useful tool to find residues that may be strained out of normal, allowed conformations, or to assess the quality of the protein structure (if 90% of the residues have phi/psi angles outside the allowed regions, you may have some doubts!). To do this you do the following: 1. In the control panel select all residues you want to see on the Ramachandran plot. 2. Choose Win-->Ramachandran Plot A Ramachandran plot will be displayed. It will look like this:

Move the mouse over the symbols on the screen to show what residues are located in what area of the plot. The residue numbe corresponding to those phi/psi angles will appear in the upper left corner of the plot. This will allow you to investigate particular residues that fall within different areas of the plot. Slab View This option, located in the Display menu allows you to slice the protein and see successive cross sections through the protein structure. When this option is checked, hold down the shift button on your keyboard and move your mouse back and forth. This will 'slice' the protein into sections that you scroll

through with your mouse. In the preferences menu you can change the width of the 'slice' that you take to allow finer and coarser views into the structure. Computing H bond distances Turning on this feature computes the H-bond distances between amino acids that are close enough to form H-bonds and have functional groups that can make these interactions. There are two things you must do to show these interactions: 1. You must compute the H-bonds. You do this by choosing Tools-->Compute H bonds 2. You must display the H-bonds by choosing Display-->Show H-bonds To show only select H-bonds you can select particular residues in the Conrol Panel or using the techniques shown below (blue table). After you do this then you: 1. Computer H-bonds by choosing Tools-->Compute H bonds 2. You display the H-bonds in a selection by choosing Display-->Show only H bonds from selection. Selecting Specific Residues There are several ways to select residues. In fact, there is a whole menu dedicated to the task of selecting particular residues. That menu is described below.

This menu offers a variety of ways to select residues. You can then decide to color these a particular color, zoom in to see these etc. Some of the most useful: None -- removes all selections. This is useful to start fresh after making some selections. All -- Selects all residues Inverse selection -- select some residues, then choose this to choose all residues but those currently selected. Visible groups -- zoom in and then use this to select those residues you have in the current view. Pick on screen -- choose then and then click on particular residue(s). These will be selected in the control panel. Group Kind -- allows you to select a particular amino acid, nucleic acid, heteroatom, solvent molecule or disulfide bond. GroupProperty -- allows you to select acidic, basic, polar and non-polar amino acids. Secondary Structure -- allows you to select all residues in a beta sheet, alph helix or coils. It also has options to select all non-trans amino acids, amino acids with phi/psi angles outside of core regions or amino acids with phi/psi angles outside of allowed regions. Several of these options are also located in the main toolbar. This will be described below. This icon, located in the main toolbar allows you to select amino acids within a defined radius . You click the icon, click on a residue on the screen and the 'Display Radius' dialog box (shown to the left) appears.

This icon doesn't do much in the way of selecting in the Control Panel, but if you click this button and then choose an atom in the structure the view will change such that the chosen atom is centered in your view.