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## Assignment 2 For DeepView Tutorial

November 30, 2001

TEACHERS: This assignment provides a means to assess your students' command of DeepView and the basics of protein structure. It is appropriate for late-first-semester biochemistry students who have completed Sections 1-11 of the DeepView Tutorial, and who have studied protein structure and some representative areas of protein function (ligand binding, catalysis).

Required for students in Biochemistry Laboratory (CHY 362) at the University of Southern Maine.

Before attempting these exercises, work through sections 1-11 of the Swiss-PdbViewer Tutorial.

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## Introduction

The following exercises introduce you to some of the advanced features of Swiss-PdbViewer. Complete all exercises and submit the required files (complete list below) by attachment to email, floppy disk (Mac or PC format), or ZIP disk (Mac format only).

### Exercise 1. Building Molecular Models to Specifications

#### Introduction

DeepView allows you to build models with specified conformations. This tutorial introduces you to tools for conformational analysis. After completing it, you will take a short segment from lysozyme and turn it into a beta hairpin.

Click on DeepView Home in the Contents frame (at left) to open the SwissPdbViewer Home Page in a new window. Click on Tutorial. Click on Making Phi/Psi statistics and carry out the tutorial. Then close the window to return to this page.

#### Exercise 1

Use what you have learned from this tutorial to build a beta hairpin structure, starting with a 17-residue sequence from lysozyme. A beta hairpin is a chain of beta structure with a beta turn in the middle. After the beta turn, the second part of the chain turns back to run parallel (antiparallel, actually) to the first portion. If you build this model well, it will contain the maximum possible number of hydrogen bonds between the first and second portions of the chain.

You need to create your own personal file for this assignment. This file will contain a short peptide sequence only -- no atomic coordinates.

Start DeepView. Open the file 1hew.pdb, in the folder hel, in the SwissPdbViewer folder (... that lay in the house that Jack built...). Use the first letter of your last name as the basis for choosing a 17-residue segment of the protein, as follows: If your last name begins with J, the 10th letter of the alphabet, select and display residues 10-26 (17 residues). Save the coordinates of this segment with the command File:Save Selected Residues. Use the name 1hew##-##.pdb, where ##-## specifies the residue range of your peptide.

Now close the file 1hew.pdb (File:Close), and open the file you just created. Save only the sequence of this segment by choosing File: Save Sequence (FASTA). Save with the name 1hew##-##.fst.

Now open your newly created FASTA file in a word processor or simple editor (like SimpleText on Macintosh), just to see what it looks like. It's little more than a string of letters: one-letter symbols for the sequence of your peptide. This is a FASTA sequence file of the type that is submitted when you search sequence databases for homologous proteins. You will conduct such a search, with a larger sequence, in the Homology Modeling exercise later. Quit your editor/word processor without saving any changes to your file.

Return to DeepView and your 17-residue segment of lysozyme. Your task (should you choose to accept it) is to use the Ramachandran diagram and commands in the Tools menu to change this structure into a hairpin: two strands of beta sheet connected by a type-II beta turn. Try to maximize the number of H-bonds between the strands. Use the Tools: Compute H-bonds command frequently as you work. DeepView does not update H-bonds when you make structural changes; you have to recompute them each time.

Here are some hints to help you. DeepView provides a command (Tools: Set Omega/Phi/Psi) for setting phi/psi angles of the selected residues to angles corresponding to common elements of secondary structure (Helix, Strand) or to prescribed values(Other...). Use this command to put your entire model into beta conformation. Then build the beta turn involving residues 8, 9, and 10 of your model.

The first residue in a beta turn has the same conformation as beta sheet. Approximate phi and psi angles for the second residue in a type-II beta turn are -60º and +120º. For the third residue, phi and psi are about +90º and 0º. Use the Tools: Set Omega/Phi/Psi:Other... command to attain these starting values. Then adjust the conformation of specific residues by clicking and dragging the corresponding dot on the Ramachandran diagram. Simplify the Ramachandran diagram by selecting only residues whose conformation you want to change.

It helps a LOT to view in stereo while adjusting conformational angles, and to orient the non-moving strand so that carbonyls lie in the plane of the screen pointing horizontally. Then move the other strand around to maximize hydrogen bonding. For now, ignore the side chains; you'll fix steric clashes after getting the main chain right.

When your model looks promising, select Tools: Energy Minimisation. This settles the chain into an energy minimum near the current conformation. The command also adds hydrogens to the model, as well as N-terminal hydrogen and C-terminal oxygen atoms, listed as the first group HHT and the last group OXT in the Control Panel. Compute H-bonds, and see whether your model needs further adjustment. If so, before resuming conformational changes, select the HHT and OXT groups and use Build: Remove Selected Residues to elmininate HHT, OXT, and as a bonus, all other hydrogens. DeepView does some wierd things when you try to move hydrogens and OXTs around. Always remove them before changing phi and psi angles or making conformational changes by other means. (If your version of DeepView does not remove all hydrogens when you remove HHT and OXT, use Build: Remove Hydrogens (All) to remove them.)

After building the turn, fix side-chain clashes in your model before saving. Find them by choosing Select:aa Making Clashes. Fix them by with Tools: Fix Selected Sidechains. Find clashes with the backbone with Select: aa Making Clashes with Backbone. You will have to fix these clashes by making small adjustments in the backbone angles (Rama diagram). (Note: terminal residues do not show up on the Rama diagram -- why not?)

Save your work as a PDB file named Hairpin.pdb (Use File:Save:Layer...). Then save a list of phi and psi angles for your model. Name the file Angles.txt. (First, select all residues; then File: Save Ramachandran Values.)

### Exercise 2. Comparing Proteins

#### Introduction

As you learned in section 11 of the tutorial, DeepView allows you to load and display more than one model at a time. This allows comparison of similar structures, and DeepView provides the means to superimpose proteins, generate structural alignments of their sequences, and to highlight vividly the similarities and differences between proteins.

If you want additional practice in superimposing proteins, click on DeepView Home in the Contents frame (at left) to open the SwissPdbViewer Home Page in a new window. Click on Tutorial. Click on Superimposing Proteins and carry out the tutorial. Then close the window to return to this page.

#### Exercise 2

Goal: To compare the apo- (protein only) and holo- (protein plus ligand) forms of a protein.

You need two models for this assignment: the model of protein-ligand complex you used in Assignment 1, and a second model of the same protein without its associated ligand (sometimes called the apoprotein, while the protein-ligand complex is called the holoprotein [Greek, from holos, whole.]). Obtain your apoprotein model from the Protein Data Bank (PDB).

Prelab preparation for this exercise will include a tour of the PDB and suggestions about how to find your apoprotein. If you cannot easily find an apoprotein model to compare with your protein-ligand complex, please ask me to help.

First, you need to change a couple of settings in SPV. Start the program and click cancel on the opening dialog box, which is for opening PDB files.

Make the following settings, using items from the Prefs menu.

1. Prefs: General
   Check the box for Scale RMS colors so that min = dark blue and max = red. This makes it easier to see slight differences between the structures of two models.
2. Prefs: Loading Protein
   Uncheck the box labeled Ignore Solvent. This allows SPV to read the coordinates of water molecules in PDB files.
   Check the box labeled Show Solvent (if loaded). This tells SPV to display water molecules, if present.

Your goal in this exercise is to create a project file in which the holoprotein (layer 2) is superimposed on the apoprotein (layer 1), with the holo form colored by RMS differences from the apo form. Blinking the layers should show conformational differences between the two forms. Save the project file with the name #xxxCompare.pdb, where #xxxx is the pdb file code of your original holoprotein model.

Study your project file:

1. Look for differences in hydrogen bonding among the residues near the ligand.
2. Look for water molecules that are displaced by the ligand when it binds.
3. Look for global differences between the models, such as relative domain movement.

Write a brief description of the conformational changes that occur when the ligand binds (no background material needed). Refer to your project file, and instruct the reader on how to see the changes you describe.

### Exercise 3. Interpreting Electron Density Maps

#### Introduction

DeepView can display electron-density maps, which are the molecular images obtained from x-ray crystallography. Scientists obtained most of the familiar protein models in your text by interpreting electron density maps. How are maps interpreted? Already knowing the amino-acid sequence of the protein (from Edman sequencing), the crystallographer displays the map with a graphics program, and then builds the molecular model within the map. In the first part of this tutorial, on fatty-acid-binding protein, you will learn how to display a map and model, how to move around in them, and how to assess the quality of the map. In the second part, on lysozyme, you will get a taste of what it's like to use a map to identify amino-acid side chains, and to adjust a model to improve its fit into the map. For this tutorial, you use a lysozyme model (called "mutant") in which four residues have been replaced by alanine. You will use the map to figure out what the residues were originally, then replace them with your guess (or better, your surmise), and fit your new side chain into the map as well as you can. Then you can compare your work with that of the crystallographer, by displaying the "correct" structure. If you are honest with yourself in carrying out this task, you will also see why you cannot make certain distinctions, such as between amino acids of the same shape, by looking at electron-density maps.

Click on DeepView Home in the Contents frame (at left) to open the SwissPdbViewer Home Page in a new window. Click on Tutorial. Click on Electron Density Maps and carry out the two tutorials provided. Then close the window to return to this page. NOTE: In the tutorials at DeepView Home, ignore all references to the command File: Reset Orientation. This command has been removed from the program and the resets are no longer necessary.

USM Students can find the required files in MACAPPS/Course Materials/BIOCHEM/SwissPdbViewer, folders fabp and hel.

#### Exercise 3

After completing the second tutorial, close the electron density map (File: Close Map). Make the mutant.pdb layer active, and use File: Save Layer to save it as a PDB file named Rebuilt.pdb. The quit from DeepView, start it again, open, in order, 1hel.pdb, the map file, and Rebuilt.pdb. If the two models are not superimposed, then use Magic Fit to superimpose Rebuilt.pdb onto 1hel.pdb (not the other way around!). Compare the models at the residues you changed. If your model does not agree with the correct model, ask yourself if it is possible to tell the difference between your choice and the "correct" answer with nothing but an electron-density map to guide you. In some cases, the answer is "no". Please do not alter your "mutant" model to agree with the correct model (if you do, you are missing the point of this exercise).

### Exercise 4. Determining a Protein Structure Using Homologous Proteins (Homology Modeling)

#### Introduction

Homology modeling is a form of structure determination of proteins, based on the assumption that proteins that are homologous in sequence are similar in structure. With nothing more than a modern computer and software, and knowing only the amino-acid sequence of a protein, you can often roughly determine its structure.

Homology modeling involves finding homologous proteins whose structure is known, and then building your sequence onto the homologous proteins as templates. This process is called threading the unknown protein onto the reference proteins. Much of threading can be automatic, but applying some judgment in regions where the protein homologies are weakest can greatly improve the model. These areas of weakest homology are usually surface loops, which require most of the manual labor of modeling. The goal in improving the loops is to align as many residues of the threaded model as possible with those of the reference proteins, and to minimize the length of gaps (visible in the model as long bonds) in regions where the model has fewer residues than the references.

After threading, the model is likely to harbor chemically unreasonable features, such as parts that clash with each other, or very long bonds across gaps. Some, but not all, clashes can be repaired with DeepView. Usually, however, the last stage of homology modeling is energy minimization, an automated process by which a computer program allows the model to settle into a lower energy conformation as similar as possible to the threaded conformation.

Two parts of homology modeling require larger and faster computers than you are likely to have on your desk: 1) the search for homologous proteins, and 2) energy minimization. For these operations, you submit the tasks to a remote computer (a server). In this tutorial, servers in Geneva, Switzerland carry out the heavy computing tasks. The pdb files of your homologous proteins come directly back to your web browser, which hands them over to DeepView. The energy minimization takes longer, and the result comes to you by e-mail. DeepView prepares both jobs for you, and submits them over the internet.

In the tutorial provided by the programmer of DeepView, the starting point is the amino-acid sequence of a protein called FASL, whose three-dimensional structure is unknown. You will find two homologous proteins and thread your model onto them, improve the fit of several loops, fix clashes, and prepare to send the model off for energy minimization. You do not need to submit the FASL model for minimization. You will do this in the exercise that follows the tutorial.

#### Exercise 4

Click on DeepView Home in the Contents frame (at left) to open the SwissPdbViewer Home Page in a new window. Click on Tutorial. Click on Homology modeling and carry out the tutorial, but do not submit a model request at the end of the tutorial. You will do this later, with a different model.

Your task in this exercise, should you choose to accept it, is to determine the structure of mouse lysozyme C. The gene for this enzyme was sequenced and posted in the mouse genome project in late 2000, but the enzyme's structure has not been determined. You will make a homology model of mouse lysozyme C, and compare its potential for binding reaction products to that of human lysozyme.

You need two files. Click the file names to obtain them: 1LZSChA and MousLysC.fst.
1LZSChA is a crystallographic model of human lysozyme complexed to (NAG)4 and (NAG)2. This is essentially an enzyme-product complex produced by adding (NAG)6 to lysozyme crystals at low temperature. The second file, MousLysC.txt, is a text file containing the sequence, in one-letter abbreviations) of amino-acid residues deduced from a gene in the mouse genome. The sequence is strongly homologous to mammalian lysozymes.

Here is an outline of what you will accomplish in this exercise. In the following paragraphs, you will find more guidance about how to carry out each step.

1. Drawing on what you learned from the homology modeling tutorial, find and use an appropriate template to make a homology model of mouse lysozyme C.
2. Submit your final model to SwissModel for optimization.
3. Compare the optimized model with the raw model.
4. Assess the capacity of the optimized model for binding the substrate NAG units that are bound to 1LZSChA.

The following instructions are not as detailed as those of a tutorial. You may have to search menus for tools you have not used before. In some cases, the point is to figure out how to get a desired result or make a desired measurement.

#### I. Making the Raw Homology Model

Start DeepView and click Cancel on the initial dialog. No windows are open, but DeepView menus are available.

SwissModel: Load Raw Sequence To Model
Browse to the file MousLys.fst and open it. It appears as a large alpha helix.

SwissModel: Find Appropriate ExPDB Templates
Your net browser (Netscape or Explorer) should start and provide a form for you to submit. Notice that your MousLys.fst sequence is being sent to a server in Geneva for a template search. ExPDB templates are single domains ro single chains extracted or exerpted from PDB files expressly for use as templates in homology modeling.

In a few seconds, a list of possible templates appears. Read the entries and find the first one that is a native model (not a mutant). As of 12/5/2000 (these databases change rapidly), the first non-mutant on the list is 1LZ1, human lysozyme. (You can read more about this model by following the link in the Parent PDB column, which takes you to the PDB and the file from which the ExPdb file is obtained.) The Blast scores tell you how closely the sequences of the templates match those of your target protein (in this case, mouse lysozyme C). Small Blast scores are better. Think of them as a probability that the match between mouse lysozyme C and the template is coincidental. The probability that the sequence correspondence between human and mouse lysozyme is a coincidence is only about 3 x 10-60. Clearly, the correspondence results from evolution, not concidence.

In the Download ExPDB column, click 1LZ1 to obtain the template. It should come to your computer and be opened by DeepView, so that now you see it sitting somewhere off the end of the MouseLys helix.

Color: Layer to give the models distinct colors.

Fit: Best (With Struct. Align)
This threads the mouse lysozyme model onto the template, and aligns their sequences. Blink between the two models to see how similar they are.

Prefs: Alignments
This preference dialog controls the appearance of sequence alignments for text files and printing. Check all boxes under "For Files:" to make a full-featured alignment file. Click OK.

On the left side of the Sequences Alignment window, click the small document icon below the ?. The alignment file appears, with the feature you set in the Alignment Preferences. It shows the two sequences aligned, with asterisks (*) marking identical residues, dots marking similar residues, and no marks under residues that are dissimilar. In addition, h and s mark residues in helical and sheet conformations.

Save: Alignment
Give the filename MouVsHum.txt. Retain this alignment file to hand in. (This file can be opened and printed in any text editor or word processor. For proper alignment, you need to apply a monospace font like Courier or Monaco.)

Wind: Sequences Alignment and Wind: Layer Infos
These two windows are handy for working with multiple models and for their sequences. Notice that homologs of some of the N-terminal residues of mouse lysozyme are not present in the human model. These residues will be missing from your new model when it returns from SwissModel.

SwissModel: Submit Modeling Request
This prepares you to send your sequence and template to the SwissModel server for automated completion of the model, which includes energy minimization. If you have not done this operation before, you will be asked for an email address. Enter an address where you can receive the final model file. It may be one to two hours before this mail comes in. Click OK and then choose SwissModel: Submit Modeling Request again, to begin a sequence of two operations:

First, you are saving your current project file. Give it the name MousLysC and save it to a convenient location. Two files will be saved there, one named MousLysC, which is only the modeling request; and a second, the project file proj_MouseLysC, which contains your work at the time of submission.

Second, you are completing the request form. Read the form carefully. Click the Browse... button and select the file proj_MousLysC. Be sure to select proj_MousLysC and NOT MousLysC. Click Open. The form returns, with the name proj_MousLysC in the submission box. Click Send Request. Retain the project file to hand in.

Now return to DeepView to explore further the raw model you have created. First, save an unaltered copy for later comparison with the final model.

Make the MousLysC layer active. File: Save: Layer
Name the file MousB4.pdb. Save it to a convenient location. This is a normal PDB file of your raw homology model.

NOTE ABOUT DeepView HANDLING OF MODELS-IN-PROGRESS: Normally, when DeepView is handling more than one model, it treats the first model as the default "reference" in all functions that require comparisons (for example, Select: aa Identical to Ref. Structure, or Color: RMS). In a homology modeling project file like proj_MousLysC, DeepView gives your model special treatment as a model-in-progress, while it treats the template (or first template is there are more than one) as the "reference" model. When DeepView opens your file MousB4.pdb, which you saved by the command File: Save: Layer, it treats it like any other PDB model, not like a model-in-progress. DeepView cannot handle two models-in-progress at the same time. To compare two models-in-progress, you must convert at least one of them the a normal PDB file using File: Save: Layer. Later in this exercise, you will compare the final and raw models, using MousB4.pdb as a normal PDB file.

Notice that the Layer Infos window has a column, Sel, that lists the number of residues selected for each model. Use this informaton and commands in the Select menu to answer these questions (in all cases there are menu commands that make it easy to answer):

Questions, Set 1.

1. How many residues does each model contain?
2. How many residues in mouse lysozyme are identical to the residues with which they are aligned in human lysozyme?
3. How many are similar?
4. How many residues in the mouse model have sidechains lacking proper H-bonds?
5. How many residues in the mouse model are making clashes with other residues? After selecting them, press return to display only the offending residues.
6. Attempt to eliminate clashes with the command Tools: Fix Selected Side Chains: Quick and Dirty. How many clashing residues remain?

The remainder of this exercise requires your final model from the SwissModel server. Stop the program and resume when the model arrives. Retain the following files: proj_MousLysC, MousB4.pdb, 1LZSChA, and MousLysC.txt.

2. Examining the Final Homology Model

You will receive several email messages, one including an attached file with a name something like "AAAa06gbN.pdb", which is a DeepView project file containing your final model and the template. Start DeepView.

File: Open PDB File...
Open the newly received project file. In it, you'll find your final homology model and the template, superimposed as they were during the work done by SwissModel. You model display includes a ribbon colored by confidence factor, a measure of how well the model fits the template. In this case, our template and target were strongly homologous, and the confidence factors are all about the same, so the ribbon is uniformly colored. If certain parts of your model could not be threaded residue-for-residue onto the template, those parts would be red.

File: Open PDB File...
Open the file MousB4.pdb. DeepView lists it at the bottom of the model shown in the Sequences Alignment and Layer Infos windows.

Answer these questions (in all cases there are menu commands that make it easy to answer):

Questions, Set 2.

1. Are there any detectable differences between the backbone conformations of MousLysC (the final homology model), and MousB4, the raw homology model? Hint: Display backbones only and blink between the two models. Use the cyc column of Layer Infos to control the models displayed during blinking.
2. Are there any detectable differences between side chain conformations of MousLysC and MousB4?
3. Does the final model contain any clashing amino acids? Compare with MousB4.
4. Does the final model contain any side chains lacking proper H-bonds? Compare with MousB4.
5. Does the final model contain any"protein problems"? Compare with MousB4.

Close MousB4.pdb.

File: Open PDB
Find and open the file 1LZSChA. Now you will compare your new model with human lysozyme with regard to its capacity of to bind the reaction products (NAG)4 and (NAG)2.

In the Sequences Alignment and Layer Infos windows, you should see the models listed in this order: MousLysC, 1LZ1, and 1LZSChA. If not, quit the program and reload models in this order.

Make 1LZSChA the active layer. Now you will superimpose 1LZSChA on your mouse model.

Fit: Best (with Struct. Align)
A dialog box asks you to specify the reference protein and the protein to align with it. Use the pop-up menus to specify MousLysC as reference, and 1LZSChA to align. Click OK. (You will see a warning that you are aligning to a model-in-progress, which is never done while building a new model, but it's OK for this comparison.) Then realign all the model sequences with Fit: Generate Structural Alignment. Now the new model and the human lysozyme/product complex are superimposed.

To assess potential NAG binding by your new model, you must merge the NAG products with the model to produce a new combined model, so that protein and NAG models are part of the same layer. Here's how:

1. Make the MousLysC layer active, and Select: All.
2. Make 1LZ1 active. Select: All and then Select: Inverse Selection. The result is that no groups are selected in 1LZ1.
3. Make 1LZSChA active. Select only the 6 residues of NAG at the bottom of the control panel.
4. Check the Sel column of the Layer Infos window to confirm that 131 groups of MousLysC, 0 groups of 1LZ1 and only 6 groups of 1LZSChA are selected.
5. Edit: Create Merged Layer From Selection.

A new layer named _merge_ appears. In the cyc column of Layer Infos, check only 1LZSChA and _merge_. Blink to compare them. Answer the following questions for both layers (in all cases there are menu commands that make it easy to answer):

Questions, Set 3.

1. How many residues are within 4.0 angstroms of the NAG groups? Don't count the NAG groups themselves.
2. How many residues make hydrogen bonds to the NAG groups? Don't count the NAG groups themselves.
3. How many of these hydrogen-bonding residues adopt significantly different side chain conformations in the two models?
4. Are the putative catalytic groups Glu35 and Asp53 in position to do their jobs in both models?

To complete this assignment, save the _merge_ layer as MousNAG6.pdb.

## File List For Assignment 2

Hand in the following files in the format preferred by your instructor (attached to e-mail, on a CD, or on a USB memory device).

Exercise-1 Files

1. 1HEW##-##.pdb: Coordinate file of your peptide from 1HEW, unmodified.
2. 1HEW##-##.fst: FASTA text file of your peptide sequence.
3. Hairpin.pdb: Coordinate file of your beta hairpin.
4. Angles.txt: Text file of phi/psi angles of your beta hairpin.

Exercise-2 Files

1. #xxxCompare.pdb: Project file comparing apo- and holo- forms of your assigned protein.
2. #xxxDescribe: Analysis of differences between apo- and holo- models.

Exercise-3 File

1. Rebuilt.pdb: Your mutant.pdb layer after replacing alanines with residues that appear to fit the electron-density map.

Exercise-4 Files

1. _proj_MousLysC: Project file as submitted to SwissModel.
2. MousB4.pdb: Raw homology model before submission to SwissModel.
3. The project file returned from SwissModel, with its original file name. This file contains the optimized model and template.
4. MousNAG6.pdb: Merged model of mouse lysozyme/NAG complex.
5. A text or word processor file with answers to question in Exercise 4, Question Sets 1-3. Explanations of answers are not necessary.

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