Molecular Modeling Lab #1: Minimizing a Polylysine Peptide with a Molecular Mechanics Force Field

Transcription

1 Molecular Modeling Lab #1: Minimizing a Polylysine Peptide with a Molecular Mechanics Force Field For this weeks report, include the following: 1) For each of these GROMACS file types, does the file contain coordinate information, topology information, both, or neither?.edr files.gro files.mdp files.top files.tpr files.trr files 2) How different is the structure of the polylysine peptide before and after the steepest descents minimization? Support your answer with a picture and a quantitative comparison between the structures. 3) Discuss how the steepest descents, conjugate gradient, and combined SD/CG minimizations differ for the polylysine peptide in a helical conformation. In your answer, be sure to include: a) the number of steps required to achieve convergence in the two methods, and b) a graph of the energy versus step number for each minimization. 4) Based on your minimizations, do you think that the polylysine peptide is more stable in the helical conformation or the second conformation (polylysine_conf2.pdb) you considered? 5) Explain why you choose to use either a steepest descents, conjugant gradient, or combined SD/CG minimization to compare the two conformations of polylysine. 6) You minimized the polylysine with two different starting conformations, and both were able to converge to a minimum energy structure. If both minimizations were considering the same peptide, why didn't they end up with the same minimum energy and structure?

2 Description of lab activities: In this lab, you will learn how to set up and perform energy minimizations of a peptide structure using the GROMACS software. We'll start by minimizing the structure of a poly-alanine helix. The overall process to set up and run a minimization is given in the flowchart below (GROMACS programs for each step in bold): Start with structure in.pdb file pdb2gmx Get a coordinate (.gro) and a topology (.top) file.mdp file with parameters for minimization protocol grompp Get a.tpr file with coordinates, topology, and parameters for minimization mdrun After completion get the following files:.log with run information.gro with final structure.edr with energies for run

3 In order to start this process, you'll have to first log in to the workstation that you'll be using this semester. You can do this using the PUTTY program. This program can be found under Start>All Programs>PUTTY>PUTTY. Upon opening this program, you'll need to enter the following information into the window: Host Name (or IP address): chemmac Choose the button for SSH Click on Open The first time you log in from a particular computer, you'll probably get a security alert. You can say Yes to this alert to log in. You will then get a new window. Enter your login and your password when prompted. Once you have logged in to your account, you will get a prompt. You are currently in your "home directory." Directories are just a different name for folders in fact, if you log on to the Macintosh machine directly you would see each directory as a folder. You can create different folders to organize your account. Let's start by making a directory for this lab by using the mkdir (make directory) command: > mkdir lab1 If you want to see that this directory has been created, you can use the ls (list) command to list the directories contained in your current directory: > ls This should list the newly created directory. You can then move into the directory using the cd (change directory) command: > cd lab1 Now, you can make a subdirectory for the first calculation you'll perform, which is a steepest descents minimization of the poly alanine structure. You can make this directory and then move into it with the following commands: > mkdir polylys_sd > cd polylys_sd

4 In order to run a minimization in GROMACS, you'll need to start with your structure. Typically, this structure is in a.pdb file. I've saved a copy of a.pdb file with the polylysine structure to my directory on the workstation. You can copy it to your account with the command: > cp /Users/instructor/modelingclass/lab1/polylys.pdb. Here the period at the end of the line copies the file to your current directory using its same name. Alternatively, you could have renamed the file with the command: cp /Users/instructor/modelingclass/lab1/polylys.pdb newname.pdb Below are the structures of a single lysine residue (on the left) and a polylysine with three residues (on the right). As you can see, lysine has an amine group in its sidechain that can be either protonated or deprotonated. The peptide you will use for simulations has 12 Lys residues in it. Now, you have the starting structure for your minimization as a.pdb file. However, you'll need to convert it into files that can be used by GROMACS. To do this, you can use a program called pdb2gmx. This will take the.pdb file and create two new files, a coordinate file (.gro) and a topology file (.top). The coordinate file will have x, y, and z coordinates for each atom of the structure in it much like a.pdb file. The topology file will contain molecular mechanics parameter information for each atom in the structure. Both of these files are necessary to run a minimization (or any other type of simulation in GROMACS).

5 You can use pdb2gmx with the following command: > pdb2gmx f polylys.pdb o polylys.gro p polylys.top inter So, what does each part of this command do? -f: This is the name of the.pdb file that you want to convert using the program -o: This is the name of the.gro (coordinate) file that will be created from the.pdb file by the program -p: This is the name of the.top (topology) file that will be created from the.pdb file by the program -inter: This command is not necessary in all cases, but I usually use it. It runs pdb2gmx in interactive mode. What this means is that the program will ask you what ionization state (protonated or not protonated) you want for all ionizable residues (Glu, Asp, Lys, and His) and whether you want charges at the N and C-termini. It will also give you options for forming disulfide bonds between Cys sidechains. If this option is not chosen, the program will default to places charges on all ionizable residues and will used charged termini; these defaults are generally acceptable in water soluble systems, but may not be ideal when dealing with membrane bound systems. You'll need to make a few choices as pdb2gmx runs. First, you'll choose a force field. You can see that GROMACS has several options, but the Gromos96 43a1 force field (option 0) is usually a good force field for proteins in aqueous solution. You'll also have to choose charges for the Lys sidechains. Let's start with a minimization with the sidechains in their protonated state (option 1). This is the charge they would typically have at a physiological ph (ph 7). You'll be asked this for each of the 12 Lys sidechains in the structure. You should also choose the options for ionized termini (- NH 4 + and COO ), which are also the typical charges at physiological conditions. (The charged termini are option 0 when asked.) After pdb2gmx completes, you can take a look at the.gro and.top files that result from it. These are just text files. You can open them up in the terminal window using a program called vi. vi is basically a very primitive word processor; there is a list of vi commands on the Unix cheat sheet I gave you. However, for just quickly looking at a file, vi can be very useful. (It is also pretty much universal on all Unix machines.)

6 Start by looking at the.gro file, using the command: > vi polylys.gro You can then use the arrow keys to scroll around the file. Using the capital G key will take you to the end of the file, and hitting a number followed by G will take you that line number. As you move through the file, you can see that the first line has a title; this is essentially blank here since there wasn't a title in my polylysine.pdb file. The second line has the number of atoms included in the file. Then, there are several lines with coordinates in them (note that the units are in nm and not Å, as in.pdb files). The last line then has information on the "box size" of the system. This isn't meaningful unless you've created a box for solvent, which we haven't done for this system. But, we'll see examples of that in the next lab. When you are finished looking at the file, you can exit vi by typing a colon (:) followed by q then RETURN. The : will take you to a "command line" at the bottom of the screen, and the q then means to quit. You can then view the topology file with the command: > vi polylys.top We won't dwell on too much on the contents of the.top file this week. But if you scroll through it you'll see that it includes information on the parameters for each atom in the structure. Other lines at the beginning say which force field is being used, and a section at the end lists what molecules are present (in this case, only a single protein molecule). Now that you have the coordinate and the topology file for the peptide you are ready to set up a minimization. To do this, you'll need one additional file that includes the parameters for the minimization, which is an.mdp file. These parameters include such things as the type of minimization to perform (e.g. steepest descents or conjugate gradient) and the maximum number of minimization steps to perform in the run. There is a sample minimization.mdp file for a steepest descents minimization saved in my directory. You can copy it to your directory using the command: > cp /Users/instructor/modelingclass/lab1/min.mdp. Let's take a look at that file to see what some of the lines mean. You can open up the file using vi with the command: > vi min.mdp

7 There are many parameters that can be used for GROMACS runs. However, in most cases you will use fairly standard values for these and you won't have to modify them significantly. On the next page I give definitions of these parameters we will focus on others as they are important over the course of the semester. For tonight, you don t really have to worry about what they mean all that much; you'll work with setting them more as the semester progresses. title=polylys_min cpp=cpp integrator=steep nsteps=1000 nstlog=10 nstenergy=5 xtc_grps=protein energygrps=protein rlist=1.0 rcoulomb=1.0 rvdw=1.0 ; This gives a title for the run ; This tells grompp the name of a "C-preprocessing" (cpp) program that it can use to interpret the.top file. This program is just called cpp on the workstation. ; This tells GROMACS to run a steepest descents minimization. ; This tells GROMACS the maximum number of steps to perform. If the minimization doesn't converge within this number of steps the run will end anyway. ; This tells GROMACS how frequently to save information to the.log file, which provides a summary of the run. ; This tells GROMACS how frequently to save energies to the energy output (.edr) file. ; This tells GROMACS which parts of the simulation system to put in a trajectory file (.xtc) that saves the coordinates during the minimization. Here, there's only a protein (e.g. no water or other molecules), so this is pretty obvious. ; This tells GROMACS which parts of the simulation system to put in a the energy output file (.edr) that saves the system energies during the minimization. Again, there's only a protein here. ; This tells GROMACS the cutoff distance (in nm) for calculating the "neighbor list" of atom pairs that need to have non-bonded interactions calculated. If this is different than the next two, it specifies a "twinrange" cutoff, where some longer range interactions (pairs > rlist but < rcoulomb or rvdw) aren't calculated every single step. ; This tells GROMACS the cutoff distance (in nm) for calculating coulombic interactions ; This tells GROMACS the cutoff distance (in nm) for calculating van der Waals interactions To set up our minimization, we need to use a program that will combine the coordinate (.gro), topology (.top), and minimization run parameter (.mdp) information into a single file (a.tpr file). This is done using the grompp program, as follows: > grompp f min.mdp o min.tpr c polylys.gro p polylys.top

8 So, what do these commands mean? -f: This is the file of minimization parameters for the run. -o: This is the output file from grompp that will be used as the start file for the actual minimization with mdrun. -c: This is the file containing the initial structure for the minimization. -p: This is the file containing the topology for the structure to be minimized. The.tpr file isn't a text file you can read. It's in machine code that will look like gibberish if you try to open it in vi. Essentially, grompp took the information from the three input files and put it into a format that is useful for GROMACS but not so accessible for humans to read! Now (finally!) we are ready to actually run our minimization. To do this, you can use the mdrun program with the following command. > mdrun v s min.tpr o min.trr x min.xtc c min.gro g min.log e min.edr So, what do these commands mean? -v: This makes the output from mdrun more verbose, which makes it clearer what the program is actually doing while it runs. This option can be omitted without interfering with the operation of the program other than decreasing the amount of output. -s: This is the start file for the run created by grompp. -o: This is the output of the trajectory of the run. It includes the coordinates for each step along the minimization. -x: This is also an output of the "trajectory" of the run, with the coordinates for each step along the minimization. However it is in a compressed format to take up less space. We'll notice more a difference between.trr and.xtc files when we perform molecular dynamics simulations next week.. -c: This is the file which will contain the final structure from the minimization in.gro format. -g: This is the log file for the job, which will include general information about how much of a run is completed (during the run) and how long the run took to complete (after the run). -e: This file contains the energies of the system throughout the run; the frequency with which it will be updated is given by nstenergy in the.mdp file.

9 The minimization should complete in a few minutes. Once it is finished, you can take a look at the.log file using vi. > vi min.log As you scroll through the file, you can see information for each step of the minimization, including the overall force field energy of the system (listed as "Potential"). Towards the bottom of the file, you can see how many steps it took to reach a converged minimum; just look for the last step that is listed with information. If the run didn't converge within the maximum number of steps, it would also tell you that. (The.log may say that the run converged to "machine precision" but not the desired forces. This is because the change in the structure and/or the energy between steps was so small it wasn't changing but that the "force" used to adjust the structure for the next step was still higher than a default threshold. For our purposes, we'll consider this to be a converged minimum.) At the bottom of the file, you can see how much time it took to complete the minimization. This time may round to zero for this very short minimization. For now, let's do two analyses of our minimization: 1) Compare the final minimized structure to the initial structure how much did it change during the minimization? 2) Look at the energies over the course of the run for this we can make a plot of potential energy versus minimization step. For the first analysis, we need to first convert our final minimized structure (saved as min.gro) to a.pdb file that we can open up and look at in SPDBV. To do this, we can use a GROMACS program called editconf. This is a program that can convert and edit coordinate files, and we'll see lots of uses for it in the coming weeks. Here, we can simply use the command: > editconf f min.gro o min.pdb So, what do these commands mean: -f: This is the input coordinate file for editconf. -o: This tells editconf what output file to save. Using the.pdb extension tells the program to save the final file in.pdb format.

10 Now, you want to download this.pdb file to the Windows computer you are working on to visualize it in SPDBV. You can do this using the WinSCP program, which you can find under Start>All Programs>WinSCP3>WinSCP. When the program opens, type the workstation name into the HostName and enter your user name and password. Also, be sure the SFTP button is chosen. If you are asked whether you wish to continue connecting and add the host name to the cache, you can say yes. You also may have to enter the password again. Once WinSCP opens, you'll have a window divided down the middle into a left and right hand side. The left-hand side shows files on your "local" computer (the Windows machine), and the right-hand side shows files on the "remote" computer (the Macintosh workstation). Navigate to the file you want to download on the right and drag it to the folder where you want it on the left. Alternatively, you can upload a file to the workstation by finding where it is saved on the local machine on the left and dragging it where you want it on the right. If you keep WinSCP open while you are working more on the workstation, it might not update the file list on the Workstation. If this happens, you can "refresh" the list of files under the Remote pull-down menu. Once the minimized structure.pdb file is on your local computer you can open it in SPDBV. (Note: When you open GROMACS.pdb files, SPDBV often thinks there is more than one model in the file although there is only one. Just tell the program to show one model.) You can also open up the initial structure for a comparison (you can either download it from the Mac workstation using WinSCP, or you can get it on the course conference). You may not be able to see both at the same time if the structure has been translated in the processing by GROMACS. However, using the fit functions, you can superimpose the structures to compare how similar or different they are. In addition to the visual comparison, you can also compare how similar they are numerically by calculating the RMS Cα deviation between the structures. (For this value, SPDBV measures the distance between the Cα positions for each of the 12 residues in the two structures. Then it computes the root mean square of these 12 values.) To calculate this value, you can select all the residues in each structure in the control panel. Then you can select the Calculate RMS option in the Fit menu, choosing the "CA (carbon alpha) only" option. The RMS value is printed in the toolbar window at the top of the screen. For the second analysis of energy at each step, you need to extract the energy values from the minimization. You could write these all down from the.log file, but that would be tedious. However, the energies were all saved in the.edr file every 10th step during the minimization. (If you look back at our run parameters we told GROMACS

11 to do this.) To extract you can use a program called g_energy. The command for this is: > g_energy f min.edr o minenergy.xvg So, what do these commands mean: -f: This is the input file with energies from the minimization for g_energy. -o: This is the file that the energies you want will be output into. (Note:.xvg files are generally a type of file you get from a variety of GROMACS analyses.) When g_energy runs, you will get lots of choices of different energy values to extract. These include the values for the different terms of the force field (e.g. bond, angle, Coulombic). Here, we're really only interested in the overall energy of the system, which is called "Potential." To select this energy, type Potential or select the corresponding number, then hit return. The program then waits for another selection. You could choose another energy term if you want. But, if you hit return again, then g_energy will begin extracting the energies from the file. Once g_energy is done, you can upload the output file onto the Windows machine using WinSCP as you did for the.pdb file. This.xvg file basically has two columns of data step number in first column and energy at that step in second column. You can make a plot of the energy versus step if you open this file in Excel. To do this, go to Open in Excel. From the "Files of type" pull down choose "All Files (*.*)". Then, open the.xvg file. This will bring up the Text Import Wizard, which will lead you through a series of steps to import the two columns into a spreadsheet. On the first step, choose "Delimited" and on the second step choose "Space" and "Treat consecutive delimiters as one." (This is because each column of data in the.xvg file is separated by several spaces.) Once you say OK to the final step, you should get an Excel file with some initial rows of general information followed by the two data columns. Once you've done this, you can make a plot of the results. Now that you've done one minimization, you can try your hand at three others. First, you can try a conjugant gradient minimization on the same polylysine peptide. You might want to perform this simulation in a new directory, or you'll need to use different names for the files. To make a new directory, you could do the following:

12 > cd.. (if you are still in the directory for the last MD run, this will take you "up" one level of directory) > mkdir polylys_cg You can now copy the files you need for the run to this directory. For this, you'll need the same polylysine.pdb file you started with last time and a.mdp file with the parameters for the conjugate gradient minimization. This file is saved as: /Users/instructor/modelingclass/lab1/min_cg.mdp And you can copy this file to your directory using a cp command analogous to those you used earlier. You can note the differences between this.mdp file and the one for steepest descents minimization: integrator=cg nstcgsteep=0 ; This says to do a conjugate gradient minimization. ; You can tell GROMACS to perform a steepest descents steep every so often during a conjugant gradient minimization. Here, we're only doing conjugant gradient steps. Second, you can do a minimization of polylysine using a conjugant gradient minimization that uses an occasional steepest descents step (every 10 steps). The.mdp file for this is saved as: /Users/instructor/modelingclass/lab1/min_combo.mdp Again, you can copy this file to your folder using cp. If you look at the.mdp file, the only different from min_cg.mdp is in the nstcgsteep=10 parameter. Third, you can do a minimization of polylysine with a different starting conformation. This will allow you to consider which conformation is more favorable for polylysine. For this, you can get a file with the structure from my directory saved as: /Users/instructor/modelingclass/lab1/polylys_conf2.pdb This can be copied to whatever directory you desire with the cp command. For this minimization, choose which of the three minimization methods (steepest descents, conjugate gradient, or a combination) you think will be best to use. You can then use the appropriate.mdp file from earlier for the method you want. Using the results from these three minimizations, you can address the questions for the weekly report.

13 Finally, when you are finished you can log out of the workstation with the following command: > logout After doing this, you can close the terminal program on your local computer.