Working with small molecules. You will use molecular modelling software to create small organic molecules and peptides, look at intercalation into DNA, explore structural similarities between molecules, and see the effects of solvents on molecular conformations.
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.
Proteins and nucleotides are not the only molecules that influence life processes. Many small molecules have a great deal of impact as well. Many hormones are small organic molecules, others are small peptides. Animal venoms are small peptides. Simple sugars fuel life processes as well as form complex structures. Many biosynthetic reactions are driven by nucleotides such as ATP.
At times the reactive nature of a large structure can be inferred by the way it reacts with smaller molecules. Large structures may require the smaller molecule in order to carry out their biological function. They may have receptor sites in which any molecule that fits the spatial conformation of the site, regardless of size, impacts the function of the site. Other small molecules may compete with required substrates to inhibit the desired reaction. Gaining knowledge about smaller molecules can lead to better understanding of the larger more complex structures.
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 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 space bar. 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 space bar.
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 space bar and then move off to the ending point of the bond, pressing the space bar 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 off 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 with 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.
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 will prompt you after every 50 passes (or iterations) to see if you want the minimization process to continue or not. This can be changed if desired.
The standard minimization run produces a molecule as it would exist in a vacuum. This is not the real world situation. 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). Water is the solvent for most, but not all life processes. Various solvent force fields have been created to produce minimization results that reflect the actual environment the molecule is in.
Exercise for week 13
This series of exercises will acquaint you with a number of different skills needed by a molecular modeller working with small molecules. These skills include: entering structural data and then minimizing it to obtain its most realistic 3-D conformation, including the effects of solvents on conformation; making physical measurements and structural comparisons on molecules; and visualizing molecules. 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.
Small molecules are deceptive. Simple compounds can and do exist in highly complex forms. Sugars for example can form extremely complex glycoproteins, the structural foundation of plants, or the protective shells of arthropods as well as provide the fuel for many life processes. Information on these molecules can be gained by examining their respective shapes, charge distributions, and solvent interactions. Color coding even small molecule structures makes the information they contain more readily apparent. What appears to be simple can be difficult to understand.
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 thirteen
Now copy over all the files needed to do this week's exercise. They are located in the directory location $UGRAD_DIR/week13.
% cp $UGRAD_DIR/week13/* .
3) Run the demo that describes this week's activities.
The demo for this week concentrates on smaller molecules. A data entry example is shown with the compound ethidium bromide. This molecule is then inserted into a DNA double strand segment and sent off to be minimized to determine the results of this insertion. Even when working with small molecules it is necessary to send jobs off to the batch queue to determine the final results.
Graphical demos run on different computer. In fact most of this week's exercise will be conducted on this machine. To reach this machine and get yourself in a directory location in an account from where you can run the demo, enter the following command.
% model1
Now get into MacroModel and view the demo for week thirteen. Entering mmv30 starts up the program. Respond to the question about a script file with week13.log and that about doing a batch process with n.
$ mmv30 week13.log n
The demo starts with entering the ethidium ion. This compound is known to intercalate into DNA. Previously there was an example of a carcinogen inserting itself into DNA. The demo moves on to show the placing of the ethidium ion into the carcinogen spot of this data set. This rough insertion attempt latter requires additional batch processing. The steps in creating an enkephalin structure is gone through. This task requires batch processing to complete as well.
ATP will be used as the molecule for the studying of solvent affects. The structure is first created from three component parts and then minimized for 100 iterations before three files are written to be used in the study. One of the data sets is sent off for normal batch processing resulting in a conformation representative of the structure in a vacuum situation. The other two use special com files that set parameters for one batch processing in a water environment and the other in chloroform setting.
Interleaved lipid molecules are looked at. An attempt is made to understand how the hydrocarbon tail sections of these molecules interact with one another. Would this type of lipid bilayer model result in structures with dimensions close in size experimentally found results? Physical measurements are made to try to find out.
The sixty carbon fullerene (the bucky ball) is looked at to explore the nature of its structure. This small molecule has generated a lot of interest. Even small molecules can contain surprises. The size of its internal cavity is looked at.
Various structure comparisons are done this week. The example contained in the demo is that of testosterone and DDE. While these molecules don't look much alike, they have similar enough characteristics to compete for the same protein receptor sites. Various approaches will be tried to determine why.
Other tasks will be carried out this week, but they are all similar in nature to the ones shown in the demo.
4) Entering a organic structure.
In this section you will use the MacroModel program to enter a small organic compound ethidium bromide. This compound is used to identify the location of DNA fragments on restriction digest gels. Long known as a powerful mutagen, it is also suspected of being a carcinogen. You will actually enter the ethidium ion, since for the purpose of this exercise the bromide ion doesn't need to be present to run the analysis process.
Use the image on the previous page as a template for the molecule to be drawn. You have been exposed to modelling enough structures by now to be able to look at such a template and come up with your own sequence of events to generate such a structure.
Points to remember. Draw in the double bonds with care. Change the necessary atoms into nitrogens. Add the plus sign, + , to the nitrogen with the ethyl group on it. When the structure matches that above, it is time to get it ready for minimization.
1) Activate MacroModel by typing mmv30.
2) Once in the program, respond to the first question by pressing the ENTER key. Respond to the prompts with 5, 4107, n, and 0.
3) When the menu window comes up, move the cursor to the DRAW button, press the space bar, move the cursor to the working window and start to enter your structure. Use your own steps to generate this molecule, keeping the points to remember in mind.
4) 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. 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. Pressing the space bar 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.
5) Minimize the structure: select ENERGY, select MM2, and then select Start when the cursor reappears. The structure entered should minimize within 200 to 300 iterations. This is a short enough period of time to sit and wait for the results.
6) Write your entered structure to a file. Select WRITE, answer the prompt for the name of file with your last name and use ethid as the extension. Press the ENTER key to the prompt for a short structure statement. The cursor will reappear when the file has been written to your account.
5) Inserting the Ethidium Ion into a DNA Segment.
1) In beginning of the course, a DNA demo showed the intercalation of a carcinogen into DNA. Later in week 9, this was explored again when the dimensions of the distorted insert site were studied. Since the ethidium ion is known to be a mutagen, is suspected of being a carcinogen, is fairly planar and is known to intercalate into DNA, it is time to model this material.
A data file containing the model DNA and carcinogen structure ready for minimization has been given the name model-hole. Read that file into the program.
2) Select the READ button. Respond to the File: prompt in the user input window with model-hole. 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 orange and red with two atoms colored blue and another two colored white.
Select the READ button again. Respond to the File: prompt with the name of your ethidium ion structure. Press the ENTER key to respond to the question about the structure number and answer with n to keep the old image on the screen. The image on the screen now shows the two data sets. Next you will remove the original carcinogen from the DNA insertion site and put in the ethidium ion.
3)To insert the ethidium ion, orient it similarly to the carcinogen already in the hole. Select INPUT, ORIENT, Mol and use the cursor to select an atom in the ethidium ion. Now select Rot, enter x for the type of rotation and -90 as the angle of rotation. Enter * to get out of doing rotations around the x axis. Enter z and use -20 as the angle of rotation. Enter * to get out of doing rotations around the z axis and then press the ENTER key to get out of the rotation mode of operation.
The image now shows the ethidium ion to the right and above the intended insert site. To move the ion in there, its old occupant needs to be removed. Select the DELT button twice to get into molecule delete mode. Move the cursor over to an atom on the carcinogen molecule and press the space bar. That molecule disappears from the screen.
Select Mol again. Use the cursor to select an atom on the ethidium ion. Select TRANS, enter y and give -4 as the distance in the y direction to move the molecule. Enter * to get out of doing translations in the y direction. Enter x and give -15 as the distance in the x direction to move the molecule. Enter * to get out of doing translations in the x direction and press the ENTER key to get out of the translation mode of operation. Your ethidium ion should now be in the insertion site area.
To get ready for the minimization process, press H ADD slowly three times. The prompt will appear, All atoms or working set (A/W)?, respond with a for all atoms. Green lines representing hydrogens will be added all over the structure.
4) Select ENERGY, to move over to the minimization portion of the program. Select It/S and enter 5 to the query about the number of iterations. To make sure that this structure will run in the batch process, select Start to test the process. If all proceeds normally for the five iterations then the batch job needed to get the final result will also run. It may take some time for the 5 passes to run, this is a relatively large structure. After they are finished, respond with n to the query about continuing and select WRITE and enter the name of your data file as insert.
It takes over 8 hours of CPU time to minimize this data. To give you a feeling for this process, a description of going through the minimization process is now given. You will not be required to do this.
In order to run a batch job you need a data file and a control file. By writing out your data to a file creates the data file. The control file is usually a modified copy of a standard com file used to run MacroModel batch jobs. The user just changes those part of the control file they need to for proper operation. In this control file, the user can select the desired name for the output file containing their results, the type of force field to be used in the calculation, and the maximum number of iterations to use. The properly modified control file is then submitted to a batch queue to be run.
The results of minimizing the ethidium/DNA insertion data is a file called insert_out. Select the READ button. Respond to the File: prompt with insert_out. Press the ENTER key to respond to the structure number question and answer with n to keep the current structure. The structure on the left is your raw data, that on the right the minimization results.
5) Check to see if the size of the hole has changed by the minimization process. Select ANALYZ. Now select Clip to zero in on the insertion region in the raw data. You will want to have the region containing the blue and white colored atoms on the screen with as little additional parts of the structure as possible. Select ADist and measure the distances between these four specially colored atoms. Record your results below.
insertion site distances: _________________________________________________
Move the cursor to Clip and press the space bar twice. The screen returns to the original two structures. This time use the Clip to zero in on the insertion region of the minimized data. Go through and measure the distances between the specially colored atoms here and record the result below.
minimized distances: _____________________________________________________
Select STOP and respond with y to the two questions asked about exiting.
6) Entering a peptide into the computer.
The last time you did this, you had to enter the amino acids that you wanted to use. This time it will only be necessary to make the peptide bonds between the desired residues. The peptide that you will be entering is the leucine form of enkephalin, YGGFL.
1) Activate MacroModel by typing mmv30. Respond to the prompts with pressing the ENTER key, 5, 4107, n, and 0.
2) Select READ. Names have been given to the amino acids you will need for this task. These names are aa_tyr, aa_gly, aa_phe and aa_leu. Enter the name for the first residue in the sequence. Once the image is on the screen select READ again. Enter the name of the second residue in the sequence. Keep the previous data on the screen.
3) Delete the bond between the carbonyl carbon and the oxygen of the hydroxyl group. Delete one of the hydrogens off the amine group of the second residue. Use the molecule deletion option of the DELT button to get rid of the free floating hydroxyl group. Draw a bond between the carbonyl carbon and the amine group using the DRAW function.
4) Select the Norml button and then use the cursor to select the nitrogen atom of the second residue's amine group. This will normalize the parameters on this atom, making a pass at fixing the very long bond distance that was just entered.
5) Select READ and enter the name of the third residue in the sequence. Again respond with n to the query about deleting the current image. Repeat the process given above for creating a peptide bond between the second and third residues in the structure. Go through this process until all the residues have been added to the structure and all their peptide bonds have been drawn and normalized.
6) Write the data to a file by selecting the WRITE button. Use enk for the filename.
It takes over 20 minutes of CPU time to minimize this data. You will not be required to do this.
The results of minimizing the enkephalin data is a file called enk_out. Select the READ button. Respond to the File: prompt with enk_out. Press the ENTER key to respond to the structure number question and answer with n to keep the current structure. The structure on the left is your raw data, that on the right the minimization results.
Look at the two structures. Record on the next page your observations on the affects of the minimization process on the raw enkephalin structure.
minimization affects: __________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________
Select STOP and respond with y to the two questions asked about exiting.
7) Generating an ATP Molecule
ATP is a small molecules that greatly affects living processes. It is composed of adenine, a ribose and a tri-phosphate unit. The energy released by cleaving off phosphate groups provides the activation energies for a large number of biological reactions.
1) Activate MacroModel by typing mmv30.
2) Once in the program, respond to the prompts by pressing the ENTER key, 5, 4107, n, and 0.
3) When the menu window comes up, move the cursor to the READ button. The components of this molecule have been given the names, ade, rib and tphos. Read in these three structures keeping the earlier one(s) each time. The final image on the screen should look like the picture given below. At this point it is necessary to delete any unnecessary atoms and make the needed bonds to link everything together.
4) Make the following bonds using the DRAW function of the program. Connect the atom labelled 1 on the adenine molecule with that of the atom labelled 2 on the ribose molecule. Then connect the atoms labelled 3 and 4 together. The hydrogen on desired hydroxyl group of the tri-phosphate molecule will be removed when you select the Updat button, so do that next.
5) Minimize the structure: select ENERGY, MM2, and then Start when the cursor reappears. This structure will take a while to minimize, so just go through 100 iterations of this process (two passes) and then stop minimizing the data. This is enough of a start to know that the minimization process works with the structure on the screen. What you really want to do with this structure is see how its conformation changes with respect to the environment it finds itself in.
6) Select WRITE. By writing the current structure to three different data files, you will have the necessary data sets to minimize the ATP raw structure into its final conformations with respect to a water, vacuum and phobic environment. Create the following three files, atp-water, atp-vac and atp-phobic. This is necessary because MacroModel locks any file that it is minimizing. Therefore, you will need three copies of the data to do the needed three minimization runs. It is always possible that your batch jobs will not be running one right after another, but rather all at the same time.
It takes from 10 minutes to over an hour of CPU time to minimize these three data sets. You will not be required to do this.
The three output data set from this minimization process are called atp-water_out, atp-vac_out and atp-phobic_out. Follow the instructions given below to work with these results.
7) Select the READ button. Read in the atp-water_out. Read in atp-vac_out, keeping the first structure. Finally read in atp-phobic_out, keeping the previous structures. Working with three structures all in default atom colors can be confusing. Color coding is just the ticket to help tell the structures apart.
8) Select ANALYZ, DISPLA, Mol, then move the cursor to select an atom on the structure on the far left. Select Mono, respond with a for the color aqua, and respond with w for working. The water result is now colored aqua. Select Mol again. This time move the cursor over to select an atom on the far right structure. Select Mono, respond with y for yellow and w for working. The images on the screen are now color coded aqua for the water results, default colors for the vacuum and yellow for the phobic results.
To really be able to compare these structures, it is necessary to have them all aligned the same way. Right now the water result has a slightly different orientation to its adenine group than the other two have. This will have to be corrected.
9) Select GEOMTR, then SuprA. Pick three sets of atom pairs. Use three of the nitrogens on the vacuum result and pair them with their corresponding nitrogens on the water result. After three atom pairs have been picked, select RigSp and respond with y to the plotting query. The water result will now be overlayed on the vacuum one. Notice how the adenine portions line up but the phosphate tails don't.
10) Move the water structure back to the left again by the following commands. Select INPUT, ORIENT, and Mol. Move the cursor over to select one of the aqua colored atoms of the phosphate tail and press the space bar. Select Trans, enter x and respond with -12<rtn> for the distance to be used. Enter *<rtn> and press the ENTER key to get out of this mode of the program's operation. The water structure is back roughly where it was before. This time its adenine portion's alignment matches the other two structures. The vacuum image is looking poorly though. Select Updat to return all the structures to sharp images.
Notice now how the phosphate tails of the three structures are all different. From this view the different environments appear to change in ribose ring, but mostly they show up in the orientation of the phosphate tail. MacroModel can create plot files of the data in the working window.
11) Select ANALYZ, then PLOT. When the cursor returns a plot file has been created.
Select STOP and respond with y to the two questions asked about exiting
You now have a file called mmod.plt in your account that contains the plotting information for the three atp structures. You will be generating an image of this over on ribozyme later in the exercise. Now, you need to ftp the data over there. This is done by following the instructions given below. Replace the expxx in the example with your actual account name. What is happening in this example is that you are logging into your ribozyme account, moving over to the thirteen subdirectory, insuring that the data transfer will be in text format and then putting the desired file in that location.
$ ftp ribozyme.vadms.wsu.edu model1.vadms.wsu.edu MultiNet FTP user process 3.4(111) Connection opened (Assuming 8-bit connections) <ribozyme.vadms.wsu.edu FTP server ready. RIBOZYME.VADMS.WSU.EDU>l expxx <Password required for expxx. Password: [enter your password] <User expxx logged in. RIBOZYME.VADMS.WSU.EDU>cd thirteen <CWD command successful. RIBOZYME.VADMS.WSU.EDU>type ascii Type: Ascii (Non-Print), Structure: File, Mode: Stream RIBOZYME.VADMS.WSU.EDU>put mmod.plt To remote file: <Opening ASCII mode data connection for 'mmod.plt'. <Transfer complete. RIBOZYME.VADMS.WSU.EDU>quit <Goodbye. $
8) Working with Lipids.
Lipids are interesting molecules. In the first week of class, a measurement on a lipid bilayer was made. It turned out to be about 64 angstroms. This was due to the way in which the model was generated with the ends of the tails up against one another instead of interleaved. This model was based on freeze fracture results. Lipid bilayers have been shown to have a range of widths depending on their physiological uses. Perhaps more thought needs to be given to the modelling of lipid bilayers. Lipids may have long hydrocarbon chains as part of their structure. To help understand lipids a good place to start is with their hydrocarbon chains.
1) Activate MacroModel with mmv30.
2) Respond to the prompts by pressing the ENTER key, 5, 4107, n, and 0.
3) Grow a hydrocarbon chain. Select GROW. Press the CH4 button 12 times. A nice section of a hydrocarbon chain appears on the screen slightly askew. Select the H ADD button and press it slowly three times. All the necessary hydrogens are added to this image. Select Rot Z and give -35 as the angle of rotation. The hydrocarbon chain is now a vertical image.
Select WRITE and create a data file called hydro. Select READ and read in that file. Don't delete the original structure. Now there are two vertical hydrocarbon chains on the screen.
4) The two chains are actually too close together. To correct this situation, select ENERGY then Dock. In response to the program prompt about selecting a molecule to dock, use the cursor to select an atom from the right-hand structure. This molecule is now the one to be docked to the original hydrocarbon chain. Pick MM2 and then Start to get the docking process underway. This goes rather quickly with these two molecules.
5) With the distance corrected, determine just how far apart the two chains are. Select ANALYZ, and then ADist. Select two corresponding atom locations on the two chains. Record this distance below. Notice how the two chains fit into one another. There is no need to repeat this measurement again as the chains are parallel to one another.
distance between the two chains: ___________________________________________
6) With data in hand for ideally fitting hydrocarbon chains, it is time to look at a lipid structure. Select READ, enter the name lipid for the file name. Don't delete the hydrocarbon chains. Select ADist again and this time select two corresponding points at the two ends of the hydrocarbon chains of the lipid. Record those values below.
left end distance between chains: __________________________________________ right end distance between chains: _________________________________________
What do the hydrocarbon portions of the lipid molecule look like? Are they parallel to one another like the hydrocarbons chains you worked with earlier? Record these observations below.
lipid molecule hydrocarbon chains alignment: ______________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________
What is the average of the two distances? Is it close to the value for the parallel chains?
average value of the two distances: _______________________________________
Now read in a file in which two lipid molecules have been interleaved together. This is to see how two lipids could possibly interact in this type of a situation.
7) Select READ, enter the name lipid-side for the file name. Delete the previous image. Select ADist again, twice. This is necessary because of a new structure being read into the program. The ADist button should go white and then green again, indicating that it is active.
Make the following measurements:
1) distance between the two blue nitrogens:________________________________ 2) distance between a blue nitrogen and the end of its saw tooth hydrocarbon chain: ___________________________________________________________________________ 3) distance between a blue nitrogen and the carbon directly across from it on the bridge between the two hydrocarbon tail sections: ___________________________________________________________________________ 4) distance between the carbon just before the ester linkage on the saw tooth hydrocarbon chain and the end of that hydrocarbon chain: ___________________________________________________________________________
Based on these measurements, what section of the lipid would have to modify its conformation in order to have this type of a structure fit within the 40 angstrom size of some lipid bilayers?
___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________
9) Looking at an unusual molecule.
Some molecules are discovered by obscure routes. Fullerenes are such molecules. They are also known as bucky balls. Fullerenes are carbon only compounds first detected in the spectra patterns of distant stars and then later in sooty candle flames. They come in all sizes, structures as small as 22 carbon atoms and up to those with well over 1000 carbon atoms. The most stable of these is a ball composed of 60 carbon atoms known as Buckminster fullerene (the bucky ball). This compound was so named after the architect Buckminster Fuller's pioneering work using geodesic domes for housing.
These interesting compounds always contain an even number of carbon atoms. Their modelled structures are composed of pentagons and hexagons. The number of hexagons increases as the number of atoms in the compound increases. When the structure gets big enough, there is a cavity inside the ball that can hold other atoms or ions. Currently work is underway with these materials for use as super conductors and as possible drug delivery systems.
1) Select READ. Enter the name sball and delete the current image on the screen. The image of the bucky ball is on the screen. MacroModel has a problem with scaling the x and y axis on these terminals. This structure really is a perfect sphere, however the image on the screen is slightly skewed in the y direction.
2) Select Clip. Move the cursor to a position on the lower left-hand potion of the working window just to the left of the bottom hexagon in the structure. Press the space bar. A red mark appears. Then move the cursor to a position just to the right of the top hexagon in the structure and press the space bar again. Your task is to cut out the middle third of the sphere. A second red mark appears and then the image is redrawn to show just the clipped section of the molecule. If your section doesn't look right, select Clip again twice to return to the original structure and try the process again.
3) Select WRITE and write out this fragment to a file called bb-ring. Respond with y to the query about saving only the displayed fragment. MacroModel has this habit of not showing a CPK model on just the atoms of a clipped structure on Tektronix terminals. It shows all the original atoms in the structure. In order to determine if there is a cavity in this structure and estimate just how big it is, only this fragment needs to be worked with.
4) Select READ and read in the data file you just wrote, bb-ring. Delete the current image on the screen. Rotate the image 90 degrees in the y direction (Rot Y, with a response of 90 to the query about the angle of rotation). The image on the screen should look like a ring. If you have any atoms projecting into the ring center, remove them. Select INPUT, DELT, and move the cursor to the atom in question and press the space bar.
5) Select ANALYZ, MODEL, and Start. A CPK version of the structure is drawn over the stick drawing. Notice the hole in the middle of the structure. How does this compare to the size of the carbon atoms that surround it? Record your observation below.
size of cavity: ____________________________________________________________ observations: ______________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________
6) Exit the program by selecting STOP and responding to the two questions with y.
10) Similar conformations?
case 1 - enkephalin and morphine
The fact that drugs from external sources can react in the brain to block out pain caused researchers to look for naturally produced pain killers. What they found were the small peptides, enkephalin [MET and LEU forms] and endorphin.
1) Activate MacroModel with mmv30.
2) Respond to the prompts by pressing the ENTER key, 5, 4107, n, and 0.
3) Select the READ button. Read in the enkephalin structure by entering enk_out. This is a minimized structure. Then read in a file containing the structure of morphine, it has the name morphine. Do not delete the first structure.
4) Color the morphine structure. Select ANALYZ, DISPLA, Mol, then move the cursor to select an atom in the morphine structure and press the space bar, Mono and give r for the color (red) and w or working. The morphine structure is now colored red.
5) Select GEOMTR. This allows molecule superpositioning. First check the distance between various points on the two molecules. Use the ADist button and selecting atoms with the cursor to do this.
On the enkephalin structure find the following distances:
1) between the oxygen on the TYR hydroxyl and the nitrogen of the amine group at the bottom of the structure ___________________________________________________________________ 2) between the same nitrogen and the oxygen of the second carbonyl group up from this point on the molecule ___________________________________________________________________ 3) between this oxygen and that of the oxygen on the TYR hydroxyl ___________________________________________________________________
On the morphine structure find these distances:
1) between the oxygen of the hydroxyl group closest to the enkephalin structure and the other hydroxyl oxygen in the structure ___________________________________________________________________ 2) between this oxygen and the nitrogen in the structure ___________________________________________________________________ 3) between the nitrogen and the oxygen of the hydroxyl group closest to the enkephalin structure ___________________________________________________________________
How do these two triangles relate to one another? Are there any sides of the triangles that are similar to one another in distance? What points on one triangle might map to points on the other one?
distance observations: _____________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________
6) Select SuprA and then select the following atom pairs. (There needs to be at least 3 sets of atom pairs to make a superpositioning possible. Orange lines will appear when a pair has been selected.)
1) the oxygen of the TYR hydroxyl group and the oxygen of the hydroxyl group on morphine closest to the enkephalin structure.
2) the bottom nitrogen on the enkephalin structure and the nitrogen on the morphine structure.
3) the carbonyl oxygen measured on the enkephalin structure and the oxygen of the morphine hydroxyl group farthest from the enkephalin structure.
7) Select RigSp. A distance of how closely the two sets of points overlapped one another will be given. Respond with y to the query about plotting the results. The results are shown on the screen with the distances still displayed.
8) To better see these results, separate the two structures. Select INPUT, ORIENT, Mol, move the cursor to an atom on the morphine structure, press the space bar, Trans, enter x, and respond to the distance query with 12 <rtn>. To the second distance prompt enter *<rtn>. Press the ENTER key again to get out of this mode of operation.
The bottom end of the two structures look similar, but not identical. They don't have to be. They just have to have similar spatial conformations in one part of the molecule to fit into the same receptor sites. Creating a Van der Waal's surface around these two molecules brings this concept into better focus.
9) Select ANALYZ, SURFAC, VDW, Rad, respond to the radius size query with .85<rtn>, Acolr, respond with a<rtn> for all surfaces, Start and respond with a<rtn> for all atoms. The images on the screen have similar spatial conformations. Morphine in this image has a sharper point than the enkephalin, but they have the same general shapes.
case 2 - testosterone and DDE
Studies have been looking for the effects of DDT on wildlife in the Florida everglades as the result of a pesticide spill in 1980. DDT's dangerous decomposition product is actually DDE. Recently it has been found that DDE appears to be competing with testosterone for the steroid receptor sites in alligators. Young male alligators have been found to have a lower hatching rate and to have very low levels of testosterone resulting in malformed cells in their testes and shrunken penises. Young female alligators have been found to have high levels of estrogen and low levels of androgens. There needs to be a balance between the sex hormones in both sexes in order to produce healthy adults. These results were determined by studying testosterone receptors and the binding of testosterone to them with and without the presence of DDE in the system.
This is another structural comparison study. Two files have been created help in this task, male for the testosterone data file and dde for the dat file on decomposition product of DDT.
1) Select the READ button. Read in the testosterone file by giving its , male, deleting the current image on the screen, and then read in the dde file. Keep the first testosterone structure on the screen. The structure on the left is testosterone and the one on the right DDE.
They don't look much alike. However, looks can be deceiving. Receptor sites are 3 dimensional structures that will accept any molecule with the spatial conformation to fit in them. They usually have some sort of charge or partial charge interaction going on to hold the desired molecule(s) in place. This requirement may be as subtle as wanting atoms with unpaired electrons at a set points in space. This distance can vary by up to an angstrom.
2) Select DISPLA, then AChrg, respond to the question about keeping the legend with n<rtn>. The structures are redrawn with a color scheme reflecting their charge distribution. They still don't look much alike. Perhaps looking at the atoms' distances might help. Select GEOMTR and then ADist. Measure the distance between the two oxygens on the testosterone molecule. Then measure the distances between the various chlorine atom combinations in the DDE structure.
testosterone distance: ___________________________________________________ Cl distances: ____________________________________________________________
Remember that the two sex hormones were almost identical in general shape and size. The only difference between the two was that the area where the carbonyl is located on testosterone was an aromatic ring with an hydroxyl group in the female hormone. Any ideas on why DDE competes with testosterone and not estradiol? Record your thoughts below.
_________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________
3) Exit the program by selecting STOP and responding to the two questions with y.
11) Looking at an Intercalated Structure.
The name of the produced output file for the ethidium/DNA insertion minimization process is insert_out. For comparison purposes this task requires working with a data file that contains a normal DNA helical segment, dna-normal, the original data used to started off the ethidium ion insertion process, model-hole and the results of the minimization, insert_out.
1) Activate MacroModel with mmv30. Respond to the prompts by pressing ENTER, 5 , 4107, n, and 0.
2) The data files you are working with are large enough so that keeping the atom labelling on the data just makes things confusing. To remove the labels select ANALYZ and then A LAB. The A LAB button will go from green to white.
3) Select the READ button. Read in the dna-normal file. Read in model-hole, keeping the first structure. Finally, read in insert_out, keeping both the previous structures. Working with three structures can be confusing. Color coding helps. The normal DNA is colored aqua and white, the other two are colored red and orange. The minimization result are on the far right.
4) Determine the distances on the normal DNA structure first (the one on the left). Select the Clip button and press the space bar. 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 normal DNA 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.
normal DNA distances:
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: ____________________________
With this information recorded, determine similar measurements on the minimized results. Select the Clip button and press the space bar twice slowly. The image on the screen returns to the original one with three DNA structures.
5)Select the Clip button and press the space bar. Move the cursor to the left and slightly below the lower blue atom of the structure in the middle (the carcinogen insert ). 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. 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. 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 and on the next page.
carcinogen insert distances:
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: ___________________________
6) Select the Clip button and press the space bar. Move the cursor to the left and slightly below the lower blue atom of the structure on the right (the minimized results). 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. 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. 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.
minimized distances:
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: ___________________________
7) Exit the program by selecting STOP and responding to the two questions with y.
12) Looking at Interesting Structures
A number of files that contain interesting structures that you can explore. To do this get back into the MacroModel program. You will be looking at three such files in this section. One is a simple organic compound, the two others are more complex structures. All have been given simple names to make this process easier.
1) Activate MacroModel with mmv30. Respond to the prompts by pressing ENTER, 5, 4107, n, and 0.
2) Select READ. Enter the name nicotine. The structure of the addictive ingredient in tobacco will be shown on the screen. Use the Rot buttons to take a closer look at this molecule. Record your impressions of the molecule below.
_________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________
3) The data files you work with next are large enough so that keeping the atom labelling on the data makes things confusing. To remove the labels select ANALYZ and then A LAB. The A LAB button will go from green to white.
4) Select READ and enter the name 1d54. Delete the previous structure. This file contains x-ray data of a drug bound to a DNA segment. One strand of the DNA is colored red, the other white. The two drug molecules are colored aqua and yellow. Use the Rot buttons to take a closer look at this molecule. Record below if you think that the drug has been intercalated into the structure or is just bound to its surface.
_________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________ _________________________________________________________________________
5) Select READ and enter the name 1dr1. Delete the previous structure. This file contains x-ray data of the protein dihdyrofolate reductase that has bound to it NADP+ and the compound biopterin. Only the protein backbone is shown in this image. The backbone is colored green, the NADP+ red and the biopterin is yellow. Use the Clip and Rot buttons to take a closer look at this molecule. Record below what sort of secondary structural elements this protein has.
________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________
What sort of secondary structural elements appear to be involved in binding the two included molecules.?
________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________
6) Exit the program by selecting STOP and responding to the two questions with y.
To get back to ribozyme for the final part of this week's exercise enter the term logout.
$ logout
13) Working with images.
The file that you ftped over here is earlier in the exercise is called mmod.plt and you will use it to generate a different type of structural image. This image will be based on the actual structure you saw in the MacroModel working window. To do this follow the instructions given below.
% gcg
% 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: *): mmod.ps<rtn>
Plotting Configuration set to:
Language: psd
Device: EPSF
Port or Queue: mmod.ps
% mmhp -out=mmod.ps
Process set to plot with EPSF attached to mmod.ps
using the psd graphic interface.
Enter file to be plotted: mmod.plt <rtn>
Enter the format wanted B&W = 0, Color = 1 : 1 <rtn>
finished with plot
%
Rename this file to reflect your lastname and then print it off on the lab's printer.
% mv mmod.ps (your lastname)-w13-m.images
% lpr (your lastname)-w13-m.images
Rename the following two Molscript files, week13-1.images and week13-2.images using the command lines given below and then print them off on the lab printer.
% mv week13-1.images (your lastname)-w13-1.images
% mv week9-2.images (your lastname)-w13-2.images
% lpr (your lastname)-w13-1.images
% lpr (your lastname)-w13-2.images
The images in week13-1.images are those of the ethidium ion on the top in two different representations, ball and stick and cpk, and testosterone and DDE in cpk representation on the bottom of the page. In week13-2.images the structure shown is that of the drug and DNA helical segment that you looked at. Add these pages to your molecular images collection.
14) 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 wee13.week13 (your lastname).week13
% pico (your lastname).week13
%rcp (your lastname).week13 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.