Gale Rhodes
Department of Chemistry
University of Southern Maine
Portland, Maine, USA 04103
rhodes@usm.maine.edu
Revised 08/05/98.
TEACHERS: See PDB File List for files appropriate for these exercises.
These exercises are required for students taking Biochemistry Laboratory (CHY362) at USM.
Assignments are arranged in order of increasing complexity, but if there are particular skills you want to develop, you can work through these assignments in any order you choose. Each assignment entails working through an advanced tutorial provided by Nicolas Guex, the creator of SPdbV, and then carrying out a related structural study. The tutorials are included in the SPdbV User Guide. You can download the PDB files called for in each tutorial from the SPdbV Home Page. (At USM, the files are located in MACAPPS/Course Materials/BIOCHEM/SwissPdbViewer/tutorialstuff.)
In each assignment, you end by saving a file that shows the results of your work. Be sure to reopen each file and make sure its contents satisfy the requirements of the assignment. Unlike RasMol files, PDB files saved with SPdbV contain both coordinates and settings, and are automatically executable -- like a combination of coordinate file and script. Just open the file, and SPdbV provides the same display you saved. Hand in all created files on a floppy disk.
In this assignment, you will study the binding of a cofactor, inhibitor, or ligand (all called hetero groups or heteromers) to a protein. With SPdbV, you can quickly find and display all neighbors to a selected group, as well as hydrogen bonds to neighbors.
Click on SPdbV Home in the Contents frame (at left) to open the SwissPdbViewer Home Page in a new window. Click on Tutorial. Click on Analysing an active site and carry out the tutorial. Then close the window to return to this page.
After you complete the tutorial, use the same PDB file I assigned you for study with RasMol (TEACHERS: See PDB File List below) to create a view that includes all residues that lie within 4.0 angstroms of the hetero group. If your pdb file contains more than one chain, restrict the view to one binding site. If your pdb file contains more than one heteromer, include only the heteromer listed in the File List. Display a dotted surface for the heteromer only.
With the model positioned to highlight the cofactor bound to its neighbors, save your work as a PDB file named CfBndg.pdb. Use File:Save As..., (NOT File:Save Selected Residues) in order to save all the coordinates. SPdbV does not save the lines showing hydrogen bonds, but you can calculate them quickly upon reloading the file.
SPdbV 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 SPdbV 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.
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 SPdbV. 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 16-residue segment of the protein, as follows: If your last name begins with J, the 10th letter of the alphabet, select and display residues 10-25 (16 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. Quit SPdbV (File:QuitB).
Double click on your newly created FASTA file, to open it with SimpleText. You have created a 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 Assignment 5 below. For now, you will use this sequence to build a beta hairpin. Quit SimpleText.
Start SPdbV by double clicking on the program icon. Click Cancel on the first dialog that appears (for opening PDB files only). Load your sequence file with this command:
SwissModel: Load Raw Sequence to Model ...
SPdbV automatically builds the sequence as an alpha helix. Your
task (should you choose to accept it) is to use the Ramachandran
diagram and commands in the Tools menu to change this helix
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.)
Here are some hints to help you. SPdbV provides a command for setting all phi/psi angles to prescribed values or to angles corresponding to common elements of secondary structure. Find this command and use it to put your 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 Phi/Psi for Selection... 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.
After building the turn, fix 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 As...). 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.)
SPdbV allows you to load and display up to 10 models at a time. This allows comparison of similar structures, and SPdbV provides the means to superimpose proteins, generate structural alignments of their sequences, and to highlight vividly the similarities and differences between proteins.
Click on SPdbV 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.
Remember that USM students can find all these files in MACAPPS/Course Materials/BIOCHEM/SwissPdbViewer/tutorialstuff.
You will need to obtain two models from the PDB for this assignment. The first, 2lzm.pdb, is the wild-type lysozyme from bacteriophage T4. The second is a mutant T4 lysozyme with one or more amino-acid replacements. The replacements were carried out in order to learn the structural effects of point mutations. I will give you a PDB file code for your very own mutant (TEACHERS: See PDB File List below). When you download it from the PDB, give it the name #xxx.pdb, where #xxx is its PDB file code.
Use SPdbV to superimpose your two models, with 2lzm as the reference protein (load it first). Determine the extent of structural changes that result from the mutation, including H-bonds present in one model but not the other. The command Color:RMS will be useful for finding and illustrating the structural differences, but some differences may be so slight that Color:RMS does not show them. To be sure that you see even slight effects of the mutation, limit the view to the mutant residue and 5 or 6 residues on either side of it. Then display the nearest neighbors of these residues in both 2lzm and your mutant. By switching between mutant and wild type, determine whether the mutation eliminates or adds any hydrogen bonds in its neighborhood.
Save at least one set of coordinates that highlight the structural differences between the mutant and wild-type lysozymes. Name the file #xxxHLT.pdb, where #xxx is the original file code for your lysozyme mutant. Write a brief description of the mutation and its effects on the structure of the enzyme. PDB information links to your mutant file will help you.
SPdbV 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 SPdbV 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.
USM Students can find the required files in MACAPPS/Course Materials/BIOCHEM/SwissPdbViewer, folders fabp and hel.
After completing the second tutorial, close the electron density map (File: Close Map). Make the mutant.pdb layer active. Use Edit:Reset Orientation to restore the model to its original orientation. Save the your modified mutant model as a PDB file named Rebuilt.pdb. (Note: If you save the model without resetting its orientation, the reloaded model will not align with the electron-density map.) Please do not alter your "mutant" model to agree with the correct model (if you do, you are missing the point of this exercise). 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".
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 SPdbV. 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 SPdbV. The energy minimization takes longer, and the result comes to you by e-mail. SPdbV prepares both jobs for you, and submits them over the internet.
In this tutorial, 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, save a copy, and send the model off for energy minimization. When you receive the minimized model, you will compare it with the saved copy to see the changes made during minimization.
NOTE: For this assignment, SPdbV needs to write files into its own folder or directory. If you are using SPdbV off a remote server (like PMLMAC.MACAPPS at USM), SPdbV cannot write files there, and will give the message, "Cannot write temporary file." To avoid this problem, before beginning this tutorial, copy the SPdbV program to your local hard disk by dragging the program icon onto your desktop. Then be sure to run this local copy of the program.
Click on SPdbV 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. You must have an e-mail account to complete the tutorial. Close the window to return to this page.
After you submit the model for optimization, make FASL the active layer. Redisplay all residues that lie in the region that is homologous to the references. Then Select: Visible Groups, and Use File: Save Selected Residues ... to save only the visible groups, thus eliminating the parts of FASL that are not homologous to the reference proteins. Save your threaded model with the name FASLBefore.pdb. After saving, but before quitting SPdbV, open the newly saved PDB file (File: Open PDB File...). Make all other layers invisible and look at FASLBefore.pdb. It should contain all residues that were homologous to the references, roughly residues 140 to 280.
You will receive the homology model by e-mail, usually within one to two hours. You will get four or more large files, the last one(s) containing the PDB coordinates for your optimized model. If your coordinate file come back in more than one piece (some mail servers break up large files into smaller ones), save them as a single file. For example, in Eudora, a widely used Macintosh e-mail utility, select all the coordinate files in the In Box window (the files may be marked 1/3, 2/3, 3/3, if broken into three files), and choose Save As... from the File menu. Do not select any of the options provided (do not include headers, do not guess paragraphs). Name the file FASLAfter.pdb and direct it to the desktop.
Now compare the threaded model (FASLBefore) with the optimized model (FASLAfter) as follows:
Start SPdbV and open FASLBefore.pdb.
Color it purple.
Load FASLAfter.pdb.
Use Tools: Magic Fit (FASLBefore as reference) to align the models.
Make FASLAfter active and use Tools: Generate Structural Alignment.
Use Tools: Fit Molecules (Auto) to improve the alignment of the two models.
Use Tools: Calculate RMS Deviation to calculate distances between corresponding groups in the two models.
Make FASLAfter the active layer and Color: RMS. The regions in which the two models are most alike are colored deep blue in FASLAfter, and other groups are colored according to their distance from the corresponding group in FASLBefore, with redder colors representing greater distances.
You can see by color in the new model where the optimization made the greatest changes.
Save the files in their current orientation, as supFASLBefore.pdb and supFASLAfter.pdb. (PC users may need to choose shorter names).
Here is a list of the files to hand in for this assignment. Due Date: TBA
Assignment 1. Analysis of Binding.
Assignment 2. Building Specified Conformations.
Assignment 3. Comparing Proteins.
Assignment 4. Interpreting Electron Density Maps.
Assignment 5. Determining a Protein Structure Using Homologous Proteins.
****
Alternative to Assignment 5, or Additional Assignment in Homology Modeling
(NOT REQUIRED OF USM STUDENTS, FALL 1997)
Teachers: If this is not enough to keep your students off the streets for a week or two, try this:
Your assignment: Using the files you were given for this assignment, ...
Provide each student with a FASTA sequence of a known (by crystallography or NMR) protein and the pdb file code of one or more known homologs; have them model the sequence on the homologs, and submit the model for energy minimization; then have them compare the minimized model with the actual structure, and comment of the strengths and weaknesses of the homology model -- will be fun to make up this assignment even if you don't use it... . Use SPdbV to make up the FASTA files: load PDB's and then save the sequence as a FASTA file.
I am looking for appropriate examples for this exercise...
TEACHERS: Here are some appropriate files for SPdbV Assignments.
For Assignment 1. Analysis of Binding
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PHTHALATE DIOXYGENASE REDUCTASE |
2PIA |
FMN |
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ADENYLATE KINASE |
1ZIN |
AP5A (ATP-analog inhibitor) |
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DNA (CYTOSINE-C5-)-METHYLTRANSFERASE |
1HMY |
S-adenosylmethionine |
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CITRATE SYNTHASE |
2CTS |
Coenzyme A |
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TRANSKETOLASE |
1TRK |
Thiamine Pyrophosphate |
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ASPARTATE AMINOTRANSFERASE |
1AAW |
Pyridoxal Phosphate |
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ADIPOCYTE LIPID-BINDING PROTEIN |
1LID |
Oleic Acid |
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AVIDIN |
1AVD |
Biotin |
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PHOSPHOENOLPYRUVATE CARBOXYKINASE |
1AYL |
ATP |
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METHIONINE SYNTHASE (B12-BINDING DOMAINS) |
1BMT |
Vitamin B12 |
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H PROTEIN OF THE GLYCINE CLEAVAGE SYSTEM |
1HPC |
Lipoic Acid |
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CELLULAR RETINOL BINDING PROTEIN |
1CRB |
Retinol |
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DIHYDROFOLATE REDUCTASE |
1DHF |
Folic Acid |
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FLAVODOXIN |
1FX1 |
FMN |
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ACETYL-COA CARBOXYLASE |
1BDO |
Biotin |
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ALLOPHYCOCYANIN |
1ALL |
Phycocyanbilin |
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HIV-1 PROTEASE |
1AJV |
Sulfamide Inhibitor AHA006 |
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HUMAN ALPHA-THROMBIN |
1LHC |
Peptide-analog inhibitor |
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DIHYDROFOLATE REDUCTASE |
1DR1 |
NADP |
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L-LACTATE DEHYDROGENASE |
1LDG |
NAD |
For Assignment 3. Comparing Proteins
Brian Matthews and his colleagues at the University of Oregon have produced many site-directed mutants of phage T4 lysozyme, in efforts to understand the effects of amino-acid substitutions on protein structure and stability. Here is a list of mutants that show structural effects sufficient to show up readily in comparisons with the wild type. Compare all mutants to the wild type, PDB file code 2lzm.
Mutations are designated by the one letter code of the wild-type residue, followed by the residue number, followed by the one letter code of the mutant residue. For example, mutant K124G contains glycine as a replacement for lysine at position 124.
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None (Wild Type) |
2LZM |
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I3C |
172L |
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I3L |
149L |
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M6I |
150L |
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S38D |
1L19 |
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P86D |
1L27 |
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R96H |
1L34 |
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Q105A |
1L00 |
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G156D |
1L16 |
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Looking for more... |
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* These file names are full of "L" and "1" (the number one), so I have put the letters into upper case to help you distinguish them. I believe that in most uses of the codes, such as searching the PDB, the case does not matter.