Working with nucleotides - section 4. You will use molecular modelling software to create nucleotide three dimensional structures, visualize the various forms of DNA, do physical measurements on nucleotide segments and explore nucleotide binding.
Author:
Susan Jean Johns
Biochemistry is the study of the molecular basis of life. All molecules have a three-dimensional conformation associated with their respective formulas. While all molecules are composed of atoms, these atoms are not rigid building blocks, but rather respond to their local environment, thereby resulting in a number of different conformations for the same formula. Working from a three-dimensional perspective can provide a great deal of insight into the chemical basis of life.
DNA is capable of forming a double stranded helix structure in which the two chains are coiled around a common axis and run in opposite directions to one another. The bases are on the inside of the helical structure. The planes of the sugars are nearly at right angles to those of the bases. The two chains are held together by hydrogen bonding between the bases. Adenine is always paired with a thymine and guanine with a cytosine.
There are a number of possible helical conformations for DNA. The standard or normal one is known as the B form. There are also an A form and a Z form. Under physiologic conditions, DNA is almost entirely in the B form. The B form contains a major and minor groove in its structure and is a right-handed double helix. In this conformation the bases have their planes perpendicular to the helical axis. The A form of DNA results from dehydrating DNA fibers below 75% humidity. This helical structure, while maintaining the right-handed nature of the B form, is shorter and wider with the base pairs tilted to the helical axis. Hairpin areas of RNA are believed to form structures similar to the A form of DNA. The Z form of DNA is a left-handed double helix. The phosphates in its backbone are zigzagged. This is the result of the repeating unit being a dinucleotide rather than a mononucleotide. Z DNA has only a single deep groove. The nature of the glycosidic bonds in the Z form alternates between anti and syn. In the A and B forms they are all anti . In Z DNA the pyrimidine base is anti and the purine base syn. Models of these different forms of DNA have been produced.
The visualization of nucleotide sequences is much more difficult than that of proteins. For one thing there are more atoms to a work with in a nucleotide than in an amino acid residue. Nucleotide sequences can be very long and even a small one pushes the limits of the modelling software. Minimization of all but the smallest sequences is out of the question.
Other things can be studied, though, like hydrogen bonding. Small segments of nucleotides can be grown in various forms. Small stem loop sections can be modelled. X-ray structures of nucleotide sequences are now starting to be determined. These are deposited in the PDB database just like protein structures. Of the approximately 4000 structures currently in PDB less than 300 are from nucleotide sources.
In order to work with a structure on the computer, its relevant information has to be entered in a form the machine can recognize. When that data doesn't exist in any other source then it is necessary for the user to input the data. Here at WSU, the VADMS Computing Resource supports the use of the MacroModel program for molecular modelling tasks. Therefore, in order to have structural data in a format that can be used by this software it should be entered either directly via the program's input mode or converted from x-ray data formats that its auxiliary software can work with. For this course all the data conversions necessary to have x-ray data available is done for you. All you have to do is enter the necessary name.
Before attempting to enter a structure into the computer with MacroModel, there are some facets of the program you should be familiar with.
1) The following is the initial screen of the MacroModel program.
When the program is started, the initial screen shows this image with the INPUT and ORGANI buttons colored green. MacroModel is set up with a working display window surrounded on the bottom and right-hand side of the screen by a series of option buttons, and an area for program messages to the user (or user input window) at the top.
The option buttons are used to communicate your wishes to the program. To activate a button, move the cursor (or cross-hair) to the button's location and press the spacebar. Cursor control on your machine is with the mouse. An activated option is colored green. The message area either informs the user of the status of an option with multiple selection possibilities, requests parameter input, or relates error messages.
2) There are two different types of option buttons, those which move the program to major functional areas, and those which set the parameters for a given function. The major function buttons are located at the bottom of the screen while parameter buttons are on the right-hand side.
3) The program assumes that all atoms drawn on the screen with the DRAW option are carbons until told otherwise. Atom types can be changed by choosing the desired atom from the list on the right-hand side of the screen, moving to the location in the structure where the change is required, and pressing the spacebar.
4) When entering double bonds with the DRAW option, it is best to choose the DRAW option again, move to the starting point of the double bond, press the spacebar and then move off to the ending point of the bond, pressing the spacebar when the cursor is in the desired position. The DRAW option is always active when the button is green, and interesting bonds can result if you do not reset the option.
5) Structures can be grown on the screen using the GROW option. Select GROW and then the unit from the listing that is desired. The purple box denotes the site of the next addition to the growing molecule. This can be changed by using the Orign button.
6) When you make a mistake and wish to remove a bond or an atom or perhaps the entire structure use the DELT button. This button can toggle three ways. Press the space bar slowly and deliberately to send the multiple signals. Pressing it once will allow the removal of a bond or an atom. Once this mode of deletion is selected, go over to the location of the atom to be removed, or the center of the bond to be removed with the cursor and press the space bar. Pressing DELT twice will allow the removal of a molecule from a screen when more than one molecule is shown. Once this mode has been selected, move the cursor over to one of the atoms of the molecule to be removed and press the space bar. The entire screen can be cleared by pressing the DELT button three times and responding with y to the question Confirm complete deletion (Y/N):.
7) The most common problem encountered in minimization (the process for turning art into a realistic structure) is forgetting to add all of the necessary hydrogens to complete the structure prior to the minimizing process. If a problem occurs during a minimization attempt, check to see that all of the hydrogens are in place. Hydrogens can be added using the H ADD button in the INPUT menu. It is necessary to select this button three times to add hydrogen to all of the molecules on the screen. A message will appear at the top of the screen stating what sort of addition is currently possible.
8) Minimization errors not cured by the addition of hydrogens require the examination of the generated mmod.err file. The information contained in this file gives the atom type, number, and type of problem encountered. To look at this file from within the program, select the TTY option, enter the term syst and then typing the mmod.err file. Viewing this file will give the numbers of the problem atoms. Enter log and press ENTER when the TTY prompt reappears to return to the program. To see how the structure is numbered, select ANALYZ and the NUM option from this menu. It may be necessary to redraw the section of the molecule where the error occurred, or parameters could be missing in the MM2 force field. If redrawing the suspect section doesn't correct the problem, contact Susan Johns for assistance.
The minimization process takes an image that has been entered into the working window of the MacroModel program and converts it into a structure in which the distances between atoms, the angles between atoms, and other structural relationships are now realistic and match that of experimentally determined parameters. This process makes use of files known as force fields that are really tables of the various parameters needed to make such calculations. A number of different force fields can be used. Each force field has strengths and weaknesses. Some are better at having the types of interactions found in organic molecules, others work best with amino acids and peptides. Experience with running such processes determines which force field to use with what type of molecule, or if a combination of force fields should be used.
Minimization insures that all the various bond lengths, bond angles, and torsional values for the molecule being processed with are within the expected parameter values. Such a process depends on the size of the molecule. The more atoms in the molecule, the longer the minimization process takes. Once a molecule reaches a certain size, say over 20 atoms or so, it is best to create a batch process to do the actual calculations and allow you to move on to other computing tasks. The standard minimization run produces a molecule as it would exist in a vacuum. Once such a structure has been created, it can then be run again with changes made in the force fields to account for the effects of solvents (i.e., its environment).
When attempting a minimization, first see if there are any problems with the structure as it appears on the screen. If there are, error messages will appear in the user input window. These need to be corrected before minimization can take place. Usually it is a simple problem like forgetting to add all the hydrogens need by the MM2 force field or not having any hydrogens for the AMBER force field. Once the error messages are corrected, the appearance of the following statement on the right hand side of the screen tells you that the minimization is beginning: Iter Movt kJ/mol. The numbers in the Iter column should get larger, those under Movt should decrease and finally reach zero, and those below kJ/mol should get as small as possible. At times this will be a negative number. The program's default settings result in you being prompted after every 50 passes (or iterations) to see if you want the minimization process to continue or not. This can be changed if desired.
Exercise for week 9
This series of exercises will acquaint you with a number of different skills needed by a molecular modeller working with nucleotide structures. These skills include: entering structural data and then minimizing it to obtain its most realistic 3-D conformation; making physical measurements on structural models; visualizing the various DNA forms; and exploring nucleotide binding. Instructions in bold should be entered followed by pressing the ENTER key. The <rtn> symbol given in program examples means to press the ENTER key as well.
Nucleotide molecular structures get complex in a hurry. The number of atoms involved in each nucleotide building block is two to three times that of an average amino acid residue. With a structural model it is possible to look at hydrogen bonding patterns. Are they the same for each of the various forms of DNA? A model allows the taking of measurements to determine the amount of distortion an intercalated molecule causes in an DNA segment. You will make measurements of this type. Color coding nucleotide structures makes the information they contain more readily apparent. What appears simple can be difficult to model.
l) Activate the computer.
Activate the machine you want to use, make connections with ribozyme and log into your account.
2) Move to this week's subdirectory and copy over to it the necessary
files.
% cd nine
Now copy over all the files needed to do this week's exercise. They are located in the directory location $UGRAD_DIR/week9.
% cp $UGRAD_DIR/week9/* .
3) Run the demo that describes this week's activities.
This week's demo deals with creating nucleotide structural data on the computer. The materials to be modelled are nucleotides - both DNA and RNA structures. The demo is a mixture of GCG information and a MacroModel presentation. The demo first determines of the secondary structure for a small segment of RNA. This is done with the GCG program FOLD. Later in the demo this RNA structure will be looked at again, this time the data is the result of minimization run on a theoretical RNA model. Various aspects of nucleotide binding are explored.
To start the first part of the demo, enter the command given below. The demo includes a MacroModel demo within it. After the first section on the RNA folding, a MacroModel session will automatically be launched. You will have to enter the name of the log file, week9.log and respond with n to the question about batch processing.
% demo9
Background information is given on the RNA folding process. A figure file is shown of the results of the folding process on the test segment of RNA. A MacroModel session is then launched and you see the automated entry of the nitrogenous base uracil. The structure is minimized.
The demo moves on to show you the automated growing of a DNA double stranded helix in the normal form. Six guanines are used to create this image. The number of hydrogen bonds in the structure is then determined. You will enter similar segments yourself in the normal and Z form and determine their respective number of hydrogen bonds. You will also explore a theoretical model of the A form.
A segment of the RNA sequence that was earlier folded by the program FOLD is next examined. RNA structures can fold and form stem loops. You will examine this model and its hydrogen bonding pattern. Only the bases from position 6 to 23 of the original sequence are contained in the model.
Chemicals can intercalate into the double stranded helix of DNA, disturbing its hydrogen bonding and changing its behavior. The demo next explores the distortion of DNA by an intercalated carcinogen, 3,4-benzpyrene. Two images appear on the screen. The one on the left is the normal DNA helical strands, the one on the right the distorted one. Atoms are color coded on each structure to aid in doing physical measurements on the size of the space between the base pairs in the middle of the displayed structures. The demo will go through this process with the normal structure. You will be doing this process on both structures in your exercise.
Chemicals can intercalate into DNA if they are flat or planar. It is hard to tell is a molecule is planar by just looking at its structure face on. However, rotating the structure in the y direction 90 degrees makes this easier to determine. You will be looking at six possible planar structures in the exercise. Working with the first one is shown here. The structure of 3,4-benzpyrene is displayed on the screen. At this point the demo will stop and you will take over. Do the following to determine if the structure is planar or not. Select Rot Y and respond with 90 to the question about the size of the angle. The image is rotated and you can see what is meant by a planar molecule, all the atoms of the molecule line up in a row. Exit the program by selecting STOP and responding with y to the two questions asked.
The demo moved you over to the modelling machine. To get back to ribozyme for the sequencing part of this week's exercise enter the term logout.
$ logout
4) Determining the secondary folding of a small RNA segment.
Little of the software contained in GCG can be used for nucleotide structural visualization. One of the few exceptions to this is the area of RNA folding. The simplest of these programs to use is FOLDRNA. By using FOLDRNA to fold up the RNA sequence and SQUIGGLES to generate a graphical output of the folded results, you can get an idea of what the folded version of the sequence looks like. FOLDRNA calculates the energy values for the folded structure based on published values for stacking and loop destabilizing energies. The program produces the optimal secondary structure for the sequence used. The resulting folded structure is actually only one of a family of structures that have the same or nearly the same energy
To explore the folding of an RNA sequence you will use a sequence that has been given the name little.rna. The sequence is small and won't take very long to fold up. Use the instructions below to carry out this task. User input is shown in bold type.
% gcg
% foldrna
FOLDRNA predicts a single optimal secondary structure for an RNA molecule
by the older method of Zuker.
FOLDRNA on what sequence ? little.rna <rtn>
What is the structure output file (* little.fld *) ? <rtn>
What is the base-by-base output file (* little.connect *) ? <rtn>
Begin (* 1 *) ? <rtn>
End (* 30 *) ? <rtn>
%
With the calculations made, move on to getting a graphical output of the results. To do this and have hard copy of the results, you will need to work with the postscript option of GCG. Using the postscript option allows you to select the type of postscript output you want. In this case VADMS has a black and white postscript printer in the teaching lab.. Enter the following commands to set up your postscript output.
% postscript
Use PostScript graphics with what device:
LaserWriter
Lzr1200
LN03-ScriptPrinter
LPS20
ColorScript-100
EPSF (single page encapsulated postscript format)<
Please choose one (* LASERWRITER *): epsf <rtn>
To what port is your EPSF connected (* /dev/ttyx *): little.ps <rtn>
Plotting Configuration set to:
Language: psd
Device: EPSF
Port or Queue: little.ps
With the output set to postscript, run the SQUIGGLES program in the following manner. Use the -sho command switch to show the base names in the structure. Use the -num=5 command switch to have the sequence numbered at multiples of five. All GCG graphical output allows the user to move the image around on the page. The default values for this particular output file puts the resulting structure on the left-hand side of the page. To move that image closer to the center, use the command switch -xpan=25. The -out=little.ps command switch is used to designate the name of the desired output file.
% squiggles -sho -num=5 -xpan=25 -out=little.ps
SQUIGGLES uses an output file from FOLDRNA to make a plot of an
RNA secondary structure.
Process set to plot with EPSF attached to little.ps
using the POSTSCRIPT graphic interface.
SQUIGGLES of what FOLDRNA output file ? little.connect
PostScript instructions for a EPSF are now being sent to little.ps.
The output file now exists. Rename the file to (your lastname)-little.ps and print it off with the commands given on the next page.
% mv little.ps (your lastname)-little.ps
% lpr (your lastname)-little.ps
Look very closely at your output. Do you notice anything unusual about the output? Record your observations below.
________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________
After you have recorded your observations, use rcp to ship this file over to the teacher account.
% rcp (your lastname)-little.ps teacher@ribozyme:receive
5) Entering nucleotide structural data into the computer.
In this section you will use the MacroModel program to enter a simple nitrogenous base structure into the computer. Move over the modelling computer.
% model1
Use this template and instructions below to help you enter the structure for the base uracil.
1) Activate MacroModel by typing mmv30.
2) Once in the program, respond to the first question by pressing the ENTER key. Then answer the question about what terminal you are using by entering 5 for a Tektronix. Enter the following responses to the asked questions with 4107, n, and 0.
3) When the menu window comes up, move the cursor to the DRAW button and press the space bar. Moving the mouse will move the cursor around on the terminal screen.
4) Put the cursor inside the working window near the left side of the screen in the middle, and press the space bar again. A letter C will appear on the screen. This point will correspond to position 1 of the template.
5) Use the cursor to move to position 2 of the template and press the spacebar again. The C will disappear and be replaced by a green line.
6) Repeat step 5 until the rest of the positions shown in the template appear on the screen. Be sure to connect positions 1 and 6 together.
7) Put in the regular double bond. Select the DRAW button. Move over to the 5 position on the structure, press the spacebar. The terminal should beep at you for finding an existing atom on the screen. Then move up to position 6 and press the spacebar again. A double bond will appear between positions 5 and 6 on the template. You must be accurate or additional atoms and not a double bond will appear on the screen.
8) Put in the carbonyl double bonds. Select the DRAW button. Move over to the 3 position on the structure, press the spacebar. The terminal should beep at you. Then move over to position 8 and press the spacebar again. Then move back to position 3 again. Another beep should result. Once this move is finished a double bond will be in the screen between positions 3 and 8. Repeat this process and generate a double bond between positions 1 and 7.
9) Now select the O from the side and move to position 7 of the structure, and press the space bar. An O should appear at the chosen site. Move over to position 8 and likewise change that carbon atom into an oxygen as well. Select the N from the side and change the atom in positions 2 and 4 into to nitrogens.
10) Now move over to the H ADD button and select it to add the required hydrogens to all of the carbons of your molecule by pressing the space bar three times, slowly. After pressing it once, hydrogen will appear on the nitrogen and the single bonded oxygen atoms. Information on the type of hydrogen addition going on will appear in the user input window. It is easy to press the keyboard keys faster than the computer can respond. This is especially true when lots of users are on the system. Pressing the spacebar slowly is the key to adding the necessary hydrogens. The user input window should have the phrase Full screen H addition at the end of this process. These hydrogens will appear as green lines off the existing atoms of the structure.
11) Minimize the structure: select ENERGY, select MM2, and then select Start when the cursor reappears. Depending on the accuracy of your initial structure, you may be asked to continue the minimization process. If asked, respond with y and keep doing so until the process stops on it own. It should prompt you to continue 2 or 3 times. You will notice that the minimization process causes the buttons on the right-hand side of the screen to change. Number are presented there to allow you to track the minimization process. The first column, Iters, should start small and go as high as needed to complete the desired process. The second column, Movt, should start high and go down to zero. The last column is the current energy value for the molecule. This should go as low as possible. Its actual value depends on the molecule being worked with. Don't be surprised to see negative values for these energy figures.
12) Write your entered structure to a file. Select WRITE, answer the prompt for the name of file with uracil, and enter a short structure statement. The cursor will reappear when the file has been written to your account.
If you wish to exit the program at this point, select STOP and respond with y to the two questions asked about exiting. Otherwise select INPUT and continue on with the exercise.
6) Entering a small nucleotide segment into the computer.
Most of the time, a user is interested in something a little bigger than a single base. Usually you are interested in a small nucleotide segment. To give you the flavor of doing this, the next section of the exercise deals with creating a DNA segment containing 8 base pairs in a double stranded helical conformation.
One problem with modelling software shows up when you attempt to create nucleotide segments. The software automatically generates some, but not all, of the known forms of DNA. Parameters in the program generate the normal (or B form) and the Z form of DNA. A theoretical model of the A form is available from the PDB database.
With this background information, create a DNA segment in the normal form using the following instructions.
section a: normal structure
1) Activate MacroModel by typing mmv30 if necessary. If you are continuing on from the previous section go to step 2. Once in the program, respond to the first question by pressing the ENTER key. Then respond to the prompts with 5, 4107, n, and 0.
2) Select the NUCLEI button. [If you are coming from the previous section then after the side buttons change, select DELT and slowly press the space bar three times. You want to clear the screen, so respond with y to the question about deleting the structure.] Now select the GROW button. After the GROW button turns green you are ready to enter your nucleotide segment. This time it will be a normal form of double stranded DNA. The necessary settings for this conformation are the defaults for the system and should already be lit, BConf, DNA and Double. For comparison's sake, your DNA segment will be composed entirely of G's on the reading strand. Move the cursor over to the Gu button and press the space bar. A pair of bases appears on the screen. The guanine is on the left-hand side of the screen. Slowly press the Gu button seven more times. You now have an 8-base pair segment of the normal form of DNA on the screen. The image shown is looking down the axis of the double stranded helix.
3) Rotate the generated structure so that you can seen the rungs of the DNA ladder. Select the Rot X button and then enter 90 for the angle of rotation. The image on the screen is now a standard representation of a DNA double stranded helix.
4) Color code the reading strand (the chain of G's). Select ANALYZ followed by DISPLA. Move the cursor to the Mol button and press the space bar. Use the cursor to select an atom on the left-hand bottom part of the structure. The terminal should beep at you when you have selected your atom. Now move the cursor to the Mono button and press the space bar. Color this side of the helical strand white. To do this answer the question about the color to use with w for white. Respond to the question about how much of the image to color with w for working set. The reading strand should now be colored white.
5) Determine the number of hydrogen bonds in this structure. Select the HBOND button. Respond to the question All atoms or working set? with a for all. The number of located hydrogen bonds will be shown in the user input window. Record that number below.
number of hydrogen bonds in the normal form of DNA: _____________________________
6) Write the data to a file by selecting the WRITE button. Use your last name for the filename and normal for the extension.
section b: Z form structure
7) Select the INPUT button to return to the data entry mode of the program. Select DELT and press the space bar three times slowly. Respond to the complete deletion question with y.
8) Select the NUCLEI button. After the side buttons change, select the GROW button. This time it will be a Z form of double stranded DNA that is created. Select the ZConf button to get the proper settings for the new segment. The rest of the settings are ok as they stand. For comparison's sake, your DNA segment will be again be composed entirely of G's on the reading strand. Move the cursor over to the Gu button and press the space bar. A pair of bases appears on the screen. The guanine is on the left-hand side of the screen. Slowly press the Gu button seven more times. You now have an 8-base pair segment of the Z form of DNA on the screen. The image shown is looking down the axis of the double stranded helix. Notice how different this image is from the previous one.
9) Rotate the generated structure so that you can seen the rungs of the DNA ladder. Select the Rot X button and then enter 90 for the angle of rotation.
10) Color code the reading strand (the chain of G's). Select ANALYZ followed by DISPLA. Move the cursor to the Mol button and press the space bar. Use the cursor to select an atom on the left-hand bottom part of the structure. The terminal should beep at you when you have selected your atom. Now move the cursor to the Mono button and press the space bar. Color this side of the helical strand aqua. To do this answer the question about the color to use with a for aqua. Respond to the question about how much of the image to color with w for working set. The reading strand should now be colored aqua.
11) Determine the number of hydrogen bonds in this structure. Select the HBOND button. Respond to the question All atoms or working set? with a for all. Record that number below.
number of hydrogen bonds in the Z form of DNA: __________________________________
12) Write the data to a file by selecting the WRITE button. Use your last name for the filename and Z form for the extension.
section c: A form structure
The MacroModel program can not create an A form of a double stranded DNA helix for you. Fortunately, a theoretical model of one exists in the PDB database. For use in this exercise this file has been given the name aform.
13) Select the READ button. Respond to the File: prompt in the user input window with aform. Press the ENTER key to respond to the question about the structure number and answer with y to delete the current image from the screen. The image on the screen is colored red and white.
14) Determine the number of hydrogen bonds in this structure. Select the HBOND button. Record that number below.
number of hydrogen bonds in the A form of DNA: ___________________________________
15) Exit the program by selecting STOP and responding to the two questions with y.
The composition of the DNA segments used to generate the normal and Z forms of DNA were the same, and so any difference in the number of hydrogen bonds found were due to differences in the forms of the helixes. This was not the case with the A form. Its composition was different and therefore the differences in the number of hydrogen bonds found could be due to other reasons.
7) Looking at RNA secondary structural elements.
In section 4 of this exercise you generated a 2-dimensional image of a folded RNA secondary structure. Now look at the modelled results of that folding. The file containing the results of the modelling effort has been given the name folded.
1) Activate MacroModel with mmv30.
2) Once in the program, respond to the first question by pressing the ENTER key. Then respond to the prompts with 5, 4107, n, and 0.
3) Select the READ button. When the File: prompt appears in the user input window, enter folded. A general stem loop structure will appear in the working window. Part of the structure is colored white. This region has the following sequence, GGACCCC. The first guanine is the farthest from the loop section.
4) Determine the hydrogen bonding pattern that the stem loop has. Select ANALYZ. From that button set, select HBOND. The user input window will report the number of hydrogen bonds found and show them in the working window as purple dashed lines. Record that number below as well as your observations on where the hydrogen bonds are located in the structure.
number of hydrogen bonds:____________________________________________________ comments on their location: _________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________ _____________________________________________________________________________
5) Exit the program by selecting STOP and responding to the two questions with y.
8) Looking at an intercalated structure.
Week one's demo on bases had an image of an intercalated carcinogen in a DNA double stranded helix. In this section you will determine just how much the normal spacing of the double stranded helix was distorted by the carcinogen. Two files have been generated for this work. The first has the name dmeasure2 and the second shole.
1) Activate MacroModel with mmv30. Once in the program, respond to the first question by pressing ENTER. Respond to the prompts with 5, 4107, n, and 0.
2) Select the READ button. When the File: prompt appears in the user input window, enter dmeasure2. An image appears with two DNA helical structures. The structure colored in aqua and white is the standard form of the helix. The one colored red and orange is the distorted one. Notice that various atoms in the two structures have been additionally color coded to make them stand out. In the regular structure the aqua strand has red atoms and the white strand green ones. The distorted structure has blue atoms in the red strand and white atoms in the orange one. Use these colored points to do measurements on the size of the spacing between a set of bases in the two structures.
3) Select ANALYZ followed by GEOMTR. You are now ready to measure the distances between the especially marked colored atoms. Select the Clip button and press the space bar. Determine the distances on the regular structure first. Move the cursor to the left and slightly below the lower red atom. Press the space bar. A red mark appears on the screen. Move the cursor to the right of the upper green atom and slightly above it. Press the space bar again. A second red mark appears and is soon replaced by the marked off section of the regular structure that fills up the working window.
Select the ADist button. Move the cursor to the lower red atom. The exact location you want is at the center of the three member star that the red color forms. Some colored stars fit this description better than others. It is the central point that you want whatever the actual image looks like. Press the space bar. Move the cursor up to the center of the top red star. Press the space bar. The distance between the two atoms is shown on the screen. Record that distance below. Repeat this process and determine the distances between the top red atom and the top green one, the top and bottom green atoms, and the bottom green and red atoms. Record all these distances below .
distance between the two red atoms: ______________________________________________ distance between the top red and green atoms: ____________________________________ distance between the two green atoms: ____________________________________________ distance between the bottom green and red atoms: _________________________________
Look closely at the shape generated by the connecting lines. How regular is its shape? Record your observations below.
comments on the generated shape from the regular helix: ________________________ ________________________________________________________________________________ ________________________________________________________________________________
With this information recorded, determine similar measurements on the distorted helix. Select the Clip button and press the space bar twice slowly. The image on the screen returns to the original one.
4) Select the Clip button and press the space bar. Move the cursor to the left and slightly below the lower blue atom. Press the space bar. A red mark appears on the screen. Move the cursor to the right of the upper white atom and slightly above it. Press the space bar again. A second red mark appears and is soon replaced by the marked off section of the distorted structure that fills up the working window.
Select the ADist button twice. Move the cursor to the lower blue atom. The exact location you want is at the center of the three member star that the blue color forms. Again some colored stars fit this description better than others. It is the central point that you want whatever the actual image looks like. Press the space bar. Move the cursor up to the center of the top blue star. Press the space bar. The distance between the two atoms is shown on the screen. Record that distance below. Repeat this process and determine the distances between the top blue atom and the top white one, the top and bottom white atoms, and the bottom white and blue atoms. Record all these distances below.
distance between the two blue atoms: ______________________________________________ distance between the top blue and white atoms: ____________________________________ distance between the two white atoms: _____________________________________________ distance between the bottom white and blue atoms: _________________________________
Look closely at the shape generated by the connecting lines. How regular is its shape? Record your observations below.
comments on the generated shape from the distorted helix: ______________________ ________________________________________________________________________________ ________________________________________________________________________________
With this data recorded, determine spacial information on the actual insertion point of the distorted structure.
5) Select the READ button. To the File: prompt, enter shole, press ENTER in response to the structure number prompt and answer with y to the deletion question.. The structure on the screen changes to a close-up of the distorted region of the red and orange structure. There is an additional structure on the screen. It is another copy of the carcinogen, this time rotated 90 degrees in the x direction to show the face on orientation of the carcinogen in the hole.
6) Select SURFAC followed by VDW. Select Rad and respond with .85 to the prompt for a surface radius. Click on the Dens button and toggle through the options until Hi density level 1 is reached. Click on AColr and then Mol. Use the cursor to select an atom in the main red colored molecular section. Then select Start. Respond to the prompt with w for working. Repeat this process to put a Van der Waals surface on the orange colored molecular section and the inserted carcinogen.
Notice that the carcinogen is planar or flat and that it fits into the space between the base pairs at an angle. The green structure on the screen is not the only planar carcinogen that is known. Molecular modelling can be used as a screening device to determine if a chemical structure is planar or not. Planar structures can insert themselves into DNA double stranded helixes, disturbing their stability.
To check out planar molecules a series of data files have been created and given the names: car0, car1, car2, car3, car4 and car5. You will look at these structures and determine if they pose a possible cancer threat by being planar or not.
7) Select the READ button, answer the File: prompt with the term car0. The image of the first structure appears in the working window. It is hard to tell if a molecule is planar by looking at the structure in this position. By rotating it in the Y direction by 90 degrees you will have a better idea if it is planar or not. Select the Rot Y button and respond to the angle question with 90. A planar structure will appear to be a thin colored vertical line. Nonplanar structures have width to them when viewed in this position. Record below whether the molecule on the screen is planar or not. Repeat this process with the remaining five structures. Recording your decision on their planarity below.
car0 is ___________ car1 is ____________ car2 is ____________ car3 is ___________ car4 is ____________ car5 is ____________
8) Exit the program by selecting STOP and responding to the two questions with y.
9) Optional (Hydrogen bonding differences due to composition changes)
The number of found hydrogen bonds varies with the composition of the nucleotide sequence. In textbooks AT pairs are said to have two hydrogen bonds between them and GC pairs three. Stacking energy is a term used to describe the connections that occur between sets of base pairs along the chain. In MacroModel there is no difference between intra and inter hydrogen bonds, all hydrogen bonds that fit the parameters of the program are reported regardless of location. To study this and possible variations based on composition, a series of files have been created for you to look at.
1) Activate MacroModel with mmv30. Once in the program, respond to the first question by pressing ENTER. Respond to the prompts with 5, 4107, n, and 0.
2) Select the READ button. When the File: prompt appears in the user input window, enter gc-at. An image appears with two DNA base pairs. The GC pair is on the left side of the screen and the AT pair is on the right. Select ANALYZ followed by HBOND. The hydrogen bonds are drawn on the screen. There are a total of eight. Note that in the GC pair that there are five instead of the reported three. This is because the modelling program works on a distance parameter. The AT pair shows three bonds instead of two. The third bond is due to an internal hydrogen bond that forms again due to the distance parameter.
3) Select the READ button again. When the File: prompt appears in the user input window, enter aa-pair. The image presented is that of two sets of base pairs shown from the side. The reading strand is AA, therefore the complement one is TT. The AA strand is on the left and the TT on the right. Select HBOND and record the number of found hydrogen bonds below. How many of these are between the stacking sets and how many between the chains?
number of stacking bonds: ____________ number of interchain bonds : ___________
Select the READ button again. When the File: prompt appears in the user input window, enter in turn each of the following names: ac-pair, ag-pair, at-pair, ca-pair, cc-pair, cg-pair, ct-pair, ga-pair, gc-pair, gg-pair, gt-pair, ta-pair, tc-pair, tg-pair and tt-pair. The image presented is that of two sets of base pairs shown from the side. The reading strand is on the left, the complement on the right. Select HBOND and record the number of found hydrogen bonds below. How many of these are between the stacking sets and how many between the chains? Repeat this process until you have data for all 16 possible base pairs.
number of hydrogen bonds found in aa-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in ac-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in ag-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in at-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in ca-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in cc-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in cg-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in ct-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in ga-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in gc-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in gg-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in gt-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in ta-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in tc-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in tg-pair: _______________________________ between chains: ___________________ between sets: _______________________ number of hydrogen bonds found in tt-pair: _______________________________ between chains: ___________________ between sets: _______________________
Based on this information, speculate on the stability of various nucleotide sequences. Which would be the most stable (have the highest number of hydrogen bonds)? Which would be the least stable (have the lowest number of hydrogen bonds)?
Most stable pair: ___________________ Least stable pair: _____________________
Generate various sequences of 4 bases in their reading strand, then determine the number of possible hydrogen bonds there would be on paper. Do this by adding the complete number of bonds for the two respective pairs and then adding in the between set number for the stacking value between the two pairs. For example, the sequence ATAT is comprised of two pairs of AT sets each with 10 hydrogen bonds, the middle pair is a TA pair that has a between sets number of hydrogen bonds of 2, the total then would be 10 + 10 + 2 or 22. This is a close estimate because the twisting of the helix will at times reduce the number of between set bonds in the top set of base pairs. Record your speculation below.
________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________
4) Exit the program by selecting STOP and responding to the two questions with y.
This finishes up the modelling part of this week's exercise, return to ribozyme for more work with nucleotide images.
$ logout
10) Working with nucleotide images.
The software that has been used in this course to create structural images was designed for generating protein images. Some types of images such as ball and stick don't depend on atom names and can be used directly on nucleotide data files. If however, a ribbon type image is wanted for the backbone portion of the nucleotide structure some trickery has to be applied to get the desired result.
The ribbon structures use the 
carbon of the protein backbone as
their tack points. These positions are approximately 3.5 angstroms apart. To
create a ribbon with a nucleotide data set requires finding atoms that are
approximately the proper distance apart and them changing their atom names to
be CA instead of their current ones.
Rename the following three Molscript files, week9-1.images, week9-2.images and week9-3.images using the command lines given below and then print them off on the lab printer.
% mv week9-1.images (your lastname)-w9-1.images % mv week9-2.images (your lastname)-w9-2.images % mv week9-3.images (your lastname)-w9-3.images % lpr (your lastname)-w9-1.images % lpr (your lastname)-w9-2.images % lpr (your lastname)-w9-3.images
The images in week9-1.images are those of two DNA double helixes. The first one is shown as a ball and stick representation and the second has the phosphate backbones of the structure displayed as ribbons. In week9-2.images the folded RNA structure you looked at before is shown. Notice the stem region of the structure. Three structures are contained in week9-3.images. They are views of the three different forms of DNA as seen looking down their helical axis. Add these pages to your growing collection of molecular images.
11) Finishing up.
Rename the report form to your last name, go into the file using the pico editor and fill it out. Rcp the form to the teacher account.
% mv week9.week9 (your lastname).week9
% pico (your lastname).week9
% rcp (your lastname).week9 teacher@ribozyme:receive
This concludes your computing session for this week. Log off ribozyme, get out of the emulator and back to the overlapping windows screen.
% logout
Press the alt and x keys together. This will cause the screen to ask if you really want to exit the program. Respond with y to get out of the teemtalk emulator and return to the overlapping windows screen.