Swiss Deep View PDF

8/2/2019 Swiss Deep View PDF

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INTRODUCTION TO SWISS DEEP VIEW:Understanding the Mechanism of a Serine Protease

References:1. Lindert, L. M. Protein structure exploration using Swiss PDB Viewer.

http://chemweb.calpoly.edu/chem/lindert/PSE/Proteinstructure-FS.htm. (11/15/04).2. Lindert, L. M. Presented at the 228th National Meeting of the AmericanChemical Society, Philadelphia, PA, August 2004; paper CHED 11.

In previous biochemistry courses you have learned to manipulate three-dimensional structures in the web-embedded molecular visualization software ProteinExplorer. The purpose of this bioinformatics exercise is to introduce an alternativemolecular visualization tool, Swiss Deep View, that offers alternative benefits/featuresto Deep View. Deep view is a free software tool like Protein Explorer, but it is not web-embedded and works well on Mac or PC platforms. PE does not. Finally, we will useDeep View at various points during the term and your efficiency with the use of theprogram is expected.

The present activity has been adapted from Dr. Lisa Lindert and will look at theprotein bovine beta trypsin. Once you have learned how to use Swiss Deep View toview structures, you will be able to download all kinds of molecular structures forvisualization/manipulation!

The free software that will be used for this exercise is available for PC or Mac at:http://www.expasy.org/spdbv/text/download.htm. You may find it helpfulthroughout the term to have this software loaded on your personal computer to allowyou to look at the structures of various biomolecules covered in the course.

Instructions for this project are shown below and are based on those found at:

.We will be following the basic instructions with modifications. See instructions below.

*if you were not aware how to reference a website, the format shown here is the AmericanChemical Society’s accepted format:

Author’s last name, first name, if available. Title of article/page as listed on the

site. URL of page. (date accessed)



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Analysis of a Serine Protease with Swiss Deep View:

Step 1: Find the Protein Data Bank entry1. Go directly to the Protein Data Bank to find our model structure for this

exercise: http://www.rcsb.org/pdb/ 2. In “Search the Archive,” type in trypsin. How many hits do you get?

What kinds of information regarding each of the structures isimmediately available? What additional information do you find whenyou hit the “Explore” button for any of the listed structures?

3. Find the PDB entry for bovine beta-trypsin, a more narrow search. Now,how many hits do you get? How could you narrow the search to bovinebeta-trypsin with an inhibitor? How many hits does that give?

4. Use the structure with PDB ID = 1TPP. The full entry for this protein will be given. What types of information are given (e.g. who were theauthors, what was the original citation, how long has this data beenposted in PDB, what was the source of the protein, etc…)? Give adescription of this entry.

5. Click the “Display Molecule” in the left side panel. The coordinates willdownload to your computer. This may take a moment.

Step 2: Load 1TPP into Deep View

1. Open the Deep View software (aka Swiss PDB-Viewer). Your screen willadjust to 250 colors.

2. In the file menu click on Open and choose the 1TPP file you justdownloaded (at this point you could open any particular structural fileyou had previously downloaded and saved, i.e. other biomolecules wemight view in this course!).

3. The structure of 1TPP should appear as a stick model. In addition, a textfile will appear on top of the program bar. Close this text window so thatwhat you see is the program bar and the protein structure in the window below the bar.

Step 3: Learning to use Deep ViewAdapted from Lindert (1):

Next we will work with this structure. Our initial goal will be the generation of a ribbondiagram and a space-filled diagram that highlights some key structural features oftrypsin. It is helpful to be familiar with the basics of the mechanism of serine proteases.Serine proteases, specifically chymotrypsin, is covered in VVP in Chapter 11, pg. 307.

These enzymes were covered in great detail in Biochemistry. As a reminder, proteaseshydrolyze amide bonds, i.e. they degrade proteins by cutting up the backbone at theamide bond. The mechanism of serine proteases involved a catalytic triad of amino acidresidues: Serine (serves as a nucleophile), Histidine (serves as an acid/base) andAspartic Acid (serves to stabilize charge on His when it is protonated on the imidazolering). While the mechanisms of all serine proteases are very similar, their specificitydiffers, that is, some cut-up proteins from the C-terminal end, some cut from the middleof a protein, some prefer to cut near bulky side chains, etc… Trypsin cleaves following



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basic amino acid residues throughout the protein. In this exercise we will look at thecatalytic triad and also examine the structure of a bound protein inhibitor in the activesite and understand why it is bound where it is bound and what aspects of the naturalsubstrate it mimics.

NOTE: Depending on the speed of your computer and its graphics capabilities you mayor may not want to toggle on the 3D rendering options in your window. To turn thesefeatures on go to the Display menu and turn on 'Render in Solid 3D' and 'Use OpenGLrendering.' 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 whenyou are finished working with the model (rotating, coloring etc.) or want to briefly seeyour model will work.

Deep View operates in as a series of windows. This is useful because you can open andclose the windows as you need them. The main toolbar will always be open. The otherwindows are opened from the Window menu located at the top right. These will openas independent windows and can be opened and closed at any time.



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The Control Panel:1. Open the Control Panel (in the main toolbar, click on “Window” and then

“Control Panel.”2. Main Features of the Control Panel are indicated and defined below:

Label ExplanationA This check-box toggles the visibility of the structure in this layerB If checked, you can move the structure aroundC The 'group' column lists the amino acid (in order from n to C terminus) with

the heteroatoms at the bottom of the list. To the left of the group colum can also be a column with letters from A-Z. If there is more than one amino acid chain ina molecule, this is shown by these letters.

D "show" is short for show backbone. If a "V" is listed (a check in Deep View) inthis column then the backbone for that particular atom will be shown

E "side" is short for side chain. If this is checked then the side chain for that

particular atom will be shown.F "labl" is short for Label. If this is checked the amino acid will be labeled in thestructure with the three letter abbreviation and the residue number. Theselabels can be changed in the Preferences menu

G This symbol stands for 'dots surface' or the van der Waals or accessible surfacearea of the protein. When checked the vdW or accessible surface will be shown.The drop down arrow allows you to select which surface will be displayed.

H "ribn" stands for Ribbon. If checked a ribbon will be traced through the backbone and it will be drawn as a helix or strand depending on theclassification of that amino acid (see K)

I 'col' stands for color. Highlighting particular residues and clicking on this box brings up a dialogue box that allows you to change the color of the selected

residue. J Drop down menu for the color option. You use this drop down menu to choose

whether you are changing the color of the backbone, backbone+sidechain,sidechain or the ribbon.

K The small letters to the left of the group name indicate whether this residue islocated in a helix or a strand. These are different from the capital letters that can be located to the left of these letters that indicate a separate chain.

Table taken from (1).



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Keystroke shortcuts for the Control Panel:clicking within a row This checks/unchecks ('V') next to a residue and

activates the feature in that columncontrol+click on a group This selects all groups that you click on while

control is pressed. This is useful for selecting agroup of residues and especially useful if they arenot continuous within the sequence.

shift+click within a column This checks/unchecks all residues in that columnwithin a layer

control+shift+click within a column This checks/unchecks all residues in that columnwithin all layers (if working with multiple layers)

Control+Z UndoTable taken from (1).

The Main Toolbar, Rotation, Zooming and Centering

The main toolbar contains all the major tools for navigating the structure, moving thestructure and changing your perspective.

Navigation, Movement and Rotation

Label DescriptionA Clicking on this button centers the molecule on the screenB Clicking on this button changes your cursor to the 'hand' which allows you to

move the entire molecule or selectionC 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 thestructure.

D Clicking on this button changes your cursor to the rotate cursor. Moving yourmouse side-to-side and up and down will rotate the molecule or selection

E Clicking on this changes the rotation from a global rotation or a rotation aboutthe axis of symmetry of the molecule. Often this is useful to play with whenyou are trying to get a particular angle and can't quite get the structure to move

the way you would like.F Toggles between moving the entire structure or just the selected residues.Table taken from (1).



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Changing Colors

You can change the color of individual residues, ribbons, loops and strucures byselecting them individually and in groups and then changing the color of the ribbon,sidechain, backbone etc. from the Control Panel. There are other options, however thatgive you important information and are easier to work with than selecting groupswithin the control panel. These selections are found in the Color drop down menu inthe Main Toolbar. The table below describes the most useful features of this menu.

In the Color Menu, use the “act on” selection at the top to toggle what is changed.

Some descriptions/definitions:Label InformationA Colors the individual atoms according to a standard color schemeB Colors by the type of amino acid. Non-polar = grey, polar =yellow, acidic =

red, basic =blue.C Colors by secondary structural element. Helices are red, sheets yellow 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.

Table taken from (1).



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Step 4: Learning more about Deep ViewAdapted from Lindert (1):

The goal of this portion of the tutorial is to get you used to some more advancedfeatures in Deep View. This will allow you to select specific residues and allow you todisplay only those residues, or to color these differently or display them in a particularrepresentation. You can also compute H-bonds to see which amino acids are connectednon-covalently. If you combine these selections, image manipulation and computationaltools with the skills you learned above you will be able to create images that convey alot of structural information.

In addition to providing a tool for viewing structures, Deep View also allows you toexamine the phi/psi angles of the residues in the protein. In the Window menu is anoption to generate a Ramachandran plot for the protein you are viewing. This is a usefultool to find residues that may be strained out of normal, allowed conformations, or toassess the quality of the protein structure (if 90% of the residues have phi/psi anglesoutside the allowed regions, you may have some doubts!).

Slab View

This option, located in the Display menu allows you to slice the protein and seesuccessive 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 thepreferences menu you can change the width of the 'slice' that you take to allow finerand coarser views into the structure.

Selecting Specific Residues

There are several ways to select residues. In fact, there is a whole menu dedicated to thetask of selecting particular residues. This menu offers a variety of ways to selectresidues. You can then decide to color these a particular color, zoom in to see these, etc.

None -- removes all selections. This is useful to start fresh after making some selections.All -- Selects all residuesInverse selection -- select some residues, then choose this to choose all residues butthose currently selected.Visible groups -- zoom in and then use this to select those residues you have in thecurrent 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 orcoils. It also has options to select all non-trans amino acids, amino acids with phi/psiangles outside of core regions or amino acids with phi/psi angles outside of allowedregions.



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Several of these options are also located in the main toolbar.

The icon with an eye and clock looking circle, located in the main toolbar, allows you toselect amino acids within a defined radius, i.e. you could find all the amino acids within

10 Å of the active site, for instance. You click the icon, click on a residue on the screenand the 'Display Radius' dialog box appears. Try it!

The icon with an eye and arrows all pointing to a central dot doesn't do much in theway of selecting in the Control Panel, but if you click this button and then choose anatom in the structure the view will change such that the chosen atom is centered in yourview. Try it!

If you would like to identify the residues located next to the inhibitor you have twooptions....

Option 1. Go to the Select menu. Click on 'Pick on Screen.' Then go on the screen andclick on the residues you wish to select. They will be highlighted in red on the controlpanel.

Option 2. Select the inhibitor heteroatom from the Control Panel. Go to the Select menu.Click on 'Neighbors of Selected aa..' then select the radius you wish to observe and theresidues within that radius will be selected. If you wish to only see these residues youmay deselect all atoms in the structure (remove all checkmarks) and then choose theoption to display only those atoms selected. This allows you to see only what is in theneighborhood of your inhibitor in the active site. Combine this with your 'Slab' viewabilities and you are dissecting an enzyme active site layer by layer!!!



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Now, with the experience using Deep View and loading a protein structure, we will begin the “real” exercise – analyzing the structure of a serine protease! Note: be sure tomaster these skills as you will see this again both on the mid-term exam and in the labportion of the course.

Project to complete:1. Create a ribbon diagram of trypsin. Color it by secondary structure and

then by secondary structure succession. Remember to turn off the sidechains.

2. Place labels at the N-terminal and C-terminal ends.

3. Make the heteroatom appear in ball and stick representation. Usecheckmarks in the “show” and “side” columns.

4. Go to “Select” menu and choose “select all.” Now, rotate the structure sothat the bound inhibitor in the active site is visible. You may need to zoom

in and out on the structure to get the best view, but keep the wholeprotein in the frame.

5. Save a copy of this view (see note below on printing). Prepare a printoutof this view with a figure caption stating what is shown.

6. Show the structure in space-fill mode, i.e. with van der Waals surface.

7. Color the residues by type. Remember to color both the back bone andside chain.

8. Keep the inhibitor in ball and stick form so it can be seen in the enzymeactive site. Make a note of the possible interactions between the inhibitorand the protein that can be seen in this view.

9. Save a copy of this view (see note below on printing). Prepare a printoutof this view with a figure caption stating what is shown.

10. Turn on “slab” mode by going into Display and clicking “slab.” Holddown “Shift” and move your mouse forward and back! Cross-sections ofthe protein now appear. Find a cross-section that clearly shows majorinteractions between the inhibitor and the residues within the active site.

11. Prepare a slab view with a figure caption stating what is shown.

12. Prepare a Ramachandran plot. What is a Ramachandran plot and whatdoes it indicate about the 1TPP protein structure?



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A note on printing from Swiss Deep View with the HHMI laptops:Once you have manipulated the structure to the desired view, press the "prtscrn"function key. The whole display is copied into the clipboard at that point. Now, pastethe image into "Paint" (which is under “Accessories”). Crop and paste the image in“Paint” to the desired region. Once you have a final version, paste from Paint intoMicrosoft Word with no distortions of the image. You may then add your captions orother embedded text easily with the image in Word and save as a Word file.

The lab report for this project should include answers to all the questions posed in boldand underlined in the instructions for the project. In addition, the following printoutsshould be included:

Printouts to attach to lab report for Deep View of a Serine Protease:- Ribbon diagram with figure caption (one page).- Space-filling model with inhibitor visible and with caption (one page).- A slab view with inhibitor visible and captions (one page).

- Ramachandran plot with caption (one page).

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