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GROMACS Introductory Tutorial
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1. Where nnname is the negative ion name CL for the Gromos G43al force field see the ions itp file for specifics wrt force field nn is the number of negative ions to add The g flag gives a name to the output log for genion When you process this command you will be prompted to provide a continuous group of solvent molecules which should be Group 12 SOL Type 12 then lt enter gt You will notice that two solvent molecules have been replaced by Cl ions Now you must make the following additions corrections to your fws top file Add include ions itp Note This should be present by default already in version 3 2 amp greater after the include statement for the force field Subtract 2 from the total number of SOL molecules and add a line for 2 Cl molecules in the molecules section at the end of the file You will also need to modify the temperature coupling scheme for your pr_md mdp and md mdp files Now you will use fws_ion pdb in place of fws_b4em pdb for generating the actual input tpr using grompp for energy minimization Temperature coupling scheme for the pr_md mdp and md mdp files after adding chloride ions Berendsen temperature coupling is on in three groups Tcoupl berendsen tau_t 0 1 0 1 0 1 tc grps protein sol CL ref t 300 300 300 Remember If you added the chloride ions you will need to run the grompp step again First remove the old fws em tpr file then run the next
2. ox fws avg pdb At the prompt for group select Group 1 Protein If you just want look at the Backbone atoms then use Group 4 Perform other analysis tasks as recommended by your instructor Appendix 2 GROMAGS Tutorial How to restart a crashed run tpbconv s prev tpr f prev trr e prev edr o restart tpr mdrun s restart tpr deffnm myrestart the deffnm option sets the default filename for all file options to mdrun How to extend a run tpbconv f traj trr s topol tpr e ener edr o tpxout tpr until SVALUE Where VALUE ps e g if you wanted to extend a 2 ns run to 5 ns then VALUE 5000 How to setup a parallel run grompp np f md mdp c fws b4md pdb r fws b4md pdb p fws top o fws md tpr Where the np flag is used to designate the number of nodes for parallel execution Then use mdrun mpi to run the parallel job For example on the UMDNJ SunFire use mprun np products gromacs sparc sun solaris2 8 bin mdrun mpi s fws md tpr o fws md trr c fws pmd pdb g md log e md edr Use the above command in your Sun Grid Engine batch submission script Bibliography l Lindahl E B Hess and D van der Spoel GROMACS 3 0 a package for molecular simulation and trajectory analysis J Mol Model 2001 7 p 306 317 2 Weber W P H H nenberger and J A McCammon Molecular Dynamics Simulations of a Polyalanine Octapeptide under Ewald Boundary Conditions Influence of Artificial Peri
3. However if you need to do more in depth analysis use make ndx to tag specific items in your model See the manual for more information How to use make ndx to setup index ndx files Use make ndx to identify particular groups that you might want to freeze during a simulation or gather special energy information Let s look at an example where we want to freeze the N and C terminal amino acids of a protein Always use make ndx to create an index group for use with the grompp program In this sample case we have a coordinate file of a collagen triple helix post position restrained dynamics where we want to freeze the N amp C terminal for production run We must first identify the residue s in the coordinate file In our case clg b4md pdb that identify the N amp C termini The command line is simple enough make ndx fclg b4md pdb o clg ter ndx You will see the following output we left out the descriptive info at the beginning followed by a command prompt gt Reading structure file Going to read 0 old index file s Analysing residue names Opening library file usr share gromacs top aminoacids dat There are 2194 OTHER residues There are 108 PROTEIN residues There are 0 DNA residues Analysing Protein Analysing Other 0 System 7365 atoms 1 Protein 765 atoms 2 Protein H H 687 atoms 3 C alpha 108 atoms 4 Backbone 324 atoms 13 GROMAGS Tutorial 5 Mainchain 435 6 MainChain Cb 5
4. 1 4 is required Under coulombtype PME stands for Particle Mesh Ewald electrostatics 4 5 PME is the best method for computing long range electrostatics gives more reliable energy estimates especially for systems where counterions like Na Cl Ca 77 etc are used Due to the nature of this particular protein having exposed charged residues with a net 2 charge to the system it is beneficial for us to use PME It is even more beneficial for us to use counterions to balance the charge and set the system to net neutral The all bonds option under constraints applies the Linear Constraint algorithm for fixing all bond lengths in the system important to use this option when dt gt 0 001 ps 6 Study the mdp file below Content of pr mdp title FWS warnings 10 cpp usr bin cpp define DPOSRES constraints all bonds integrator md dt 0 002 ps nsteps 10000 total 20 0 ps nstcomm 1 nstxout 250 collect data every 0 5 ps nstvout 1000 nstfout 0 nstlog 10 nstenergy 10 nstlist 5 ns_type grid rlist 0 9 coulombtype PME rcoulomb 0 9 rvdw 1 4 fourierspacing 0 12 fourier nx 0 fourier ny 0 fourier nz 0 pme_order lt 4 ewald rtol de 5 optimize fft yes Berendsen temperature coupling is on in two groups Tcoupl berendsen tau_t u E 0 1 0 1 use items in bold if you have Cl ions tc grps protein sol CL ref t 300 300 3
5. Descents in vacuo A User kerrigan 236 ji Input file cpp usr bin cpp constraints none integrator steep nsteps 400 Energy minimizing stuff emtol 1000 emstep 0 01 nstcomm l ns_type grid morse no coulombtype Shift vdw_type Shift 18 GROMACS Tutorial rlist rcoulomb rvdw rcoulomb switch rvdw switch epsilon r Tcoupl Pcoupl no gen vel no oaorrrr rr DVOoNNih ll S3 O Conjugate gradient with morse potential ra in vacuo User kerrigan 236 Input file cpp usr bin cpp constraints none integrator cg nsteps 3000 A Energy minimizing stuff emtol 100 emstep 10201 nstcgsteep 1000 nstcomm l morse yes coulombtype Shift vdw_type Shift ns_type grid rlist 1 4 rcoulomb B rvdw rcoulomb switch rvdw_switch epsilon r orrrr SOON Tcoupl no Pcoupl no gen vel no g hbond Use g hbond to measure the number of hydrogen bonds hbond distances amp angles between molecules or groups through the course of the simulation g hbond f fws md trr s fws md tpr num fws hnum xvg 19 GROMACS Tutorial Geometrical relations used by g hbond The defaults in Gromacs 3 2 are r lt 0 35nm a lt 30 Use the r and a flags to set other limits By default g hbond analyzes the donor acceptor rpa distance You may change this behaviour by setting the da flag t
6. an output file fit pdb The output file will show the two structures superimposed on each other g covar Use to compute the covariance matrix see manual for more info May also be used to compute an average structure from a dynamics trajectory For example to compute the average of the last 200 ps of a 1 ns dynamics run use g covar f traj xtc s topol tpr b 801 e 1000 av traj avg pdb Warning Average structures tend to be crude Minimization is recommended 15 GROMAGS Tutorial g energv Use this program to plot energy data pressure volume density etc g energy f md edr o fws pe xvg You will be prompted for the specific data that you want to include in the output file xvg The output file is a type of spreadsheet file that can be read using Xmgr or Grace This file is a text file and can be read into Microsoft Excel however you will need to do some light editing of the file For example in our case above we see the following Select the terms you want from the following list G96Angle Proper Dih Improper Dih LJ 14 Coulomb 14 LJ SR LJ LR Coulomb SR Coul recip Potential Kinetic En Total Energy Temperature Pressure bar Box X Box Y Box Z Volume Densitv SI pv Vir XX Vir XY Vir XZ Vir YX Vir YY Vir YZ Vir ZX Vir ZY Vir Z4Z Pres XX bar Pres XY bar Pres XZ bar Pres YX bar Pres YY bar Pres YZ bar Pres ZX bar Pres ZY bar Pres Zz bar Surf Su
7. grompp command below We added chloride ions in our model to neutralized the overall net charge of the model grompp f em mdp c fws 1on pdb p fws top o fws em tpr Run the energy minimization in background Use the ampersand amp at the end of the command mdrun s fws em tpr o fws em trr c fws b4pr pdb g em log e em edr amp Use the tail command to check on the progress of the minimization tail 15 em log When the minimization is complete you should see the following summary in your log file indicating that steepest descents converged Use tail 50 em log Steepest Descents converged to Fmax 1000 in 207 steps Potential Energy 1 8840495e 05 Maximum force 9 8538837e402 on atom 109 Norm of force 5 3166636e 03 GROMACS Tutorial Setup the position restrained molecular dynamics What is position restrained MD You are restraining or partially freezing if you will the atom positions of the macromolecule while letting the solvent move in the simulation This is done to soak the water molecules into the macromolecule The relaxation time of water is 10 ps Therefore we need to do dynamics in excess of 10 ps for position restrained work This example uses a 20 0 ps time scale at least an order of magnitude greater The settings below will work well for the Gromacs force field Consult the Gromacs manual for optimal settings for other force fields e g the GROMOS 96 force field uses nstlist 5 and rvdw
8. group name nr name splitch nr Enter list groups a atom amp del nr splitres nr l list residues t atom type keep nr splitat nr h help r residue res nr chain char name group case case sensitive q save and quit gt We used the v command to verify that our name change was successful To finish up use the q command to save and quit and you re done Now how do we freeze the groups Easy add the following lines to your md mdp file energygrps excl Terminal Terminal Terminal SOL To remove computation of nonbonding interactions between the frozen groups with each other and surroundings i e the solvent SOL freezegrps Terminal Index group to freeze freezedim gt Ysy Y Freeze this group in all directions x y and z Remember you must include the new index file when you use this md mdp file to create your run input file tpr using grompp Use the n flag to grompp For example grompp f md mdp c clg b4md pdb p clg top n clg ter ndx o clg md tpr Properties g confrms To compare the final structure to the original PDB file obtained from the PDB use g confrms for a description use g confrms h This program takes two structures and performs a least squares fit g_confrms f1 IOMB pdb f2 fws pmd pdb o fit pdb You will be prompted to select a group select the Backbone group 4 both times The program reports an RMS deviation and produces
9. to view the trajectory you may also download to your PC and use VMD to view the trajectory ngmx f fws md trr or fws md xtc s fws md tpr When the viewer comes up you will see a dialog box with a number of selections Check the box labeled Protein then click Ok 10 GROMAGS Tutorial FUS TOXINr in water I ve Got Two Turntables and a Microphone B Hansen Group 1 Sustem Protein OProtein H C alpha Backbone OMainChain O ainChaintCb ainChaintH SideChain OSideChain H OProt asses Non Protein SOL QOther Ok ngmx s initial startup dialog 11 lt Y Hove gt lt 2 Hove gt lt Scale gt GROMAGS Tutorial File Displa Help FUS TOXINr in water I ve Got Two Turntables and a Microphone B Hansen lt X Rotate gt lt Y Rotate gt lt 2 Rotate gt lt X Move gt lt Y Hove gt lt 2 Move gt lt Scale gt Selecting Protein permits us to view the protein only without interference from the 3 000 water molecules that occupy the rest of the box Use X Rotate to rotate the box up and down left mouse click goes up right mouse click goes down Use Y Rotate to rotate the box from left to right or vice versa left mouse click rotates to the left and right mouse click rotates to the right Scale at the bottom permits you to zoom in and zoom out use left mouse button to zoom in and r
10. 0 g rms s fws md tpr f fws md trr o fws rmsd xvg Select group 4 backbone for the least squares fit The program will generate a plot of rmsd over time rmsd xvg which you may import into Excel as a space delimited file g rmsf Computes root mean square fluctuation of atom positions Like g covar this command may be used to compute average structures and may be faster than g covar For example to calculate the average of the last 500 ps of a 2 ns 2000 ps dynamics simulation use the following g rmsf f traj xtc s topol tpr b 1501 e 2000 ox traj avg pdb Select a range where you observe some equilibrium from your RMSD plot computed using g rms For example 17 GROMACS Tutorial RMSD Backbone after Isq fit to Backbone 0 2 T T T 0 15 RMSD nm e 0 05 l 0 50 100 Time ps In the example above we might use the 30 to 90 ps range for computing an average structure as we observe a leveling off in deviation from the reference initial structure Warning Average structures tend to be crude Minimization is recommended Suggested em mdp files setup for an in vacuo minimization Perform steepest descents first followed by conjugate gradient CAUTION You may need to run pdb2gmx to setup a new set of topology files for your minimization especially if you have isolated a specific group in computing your average structure as opposed to the entire system Steepest
11. 00 Pressure coupling is on GROMAGS Tutorial Pcoupl berendsen tau_p 05 compressibility 4 5e 5 ref p 1 0 Generate velocites is on at 300 K gen vel yes gen temp 300 0 gen_seed 173529 Important things to know about the mdp file The DPOSRE in the define statement tells Gromacs to perform position restrained dynamics The constraints statement is as previously discussed all bonds sets the LINCS constraint for all bonds 6 The integrator tells gromacs what type of dynamics algorithm to use another option is sd for stochastic dynamics dt is the time step we have selected 2 fs however this must be entered in units of ps nsteps is the number of steps to run just multiply nsteps X dt to compute the length of the simulation nstxout tells gromacs how often to collect a snapshot for the trajectory e g nstxout 250 with dt 0 002 would collect a snapshot every 0 5 ps coulombtype selects the manner in which Gromacs computes electrostatic interactions between atoms PME is particle mesh ewald there are other options like cut off rcoulomb and rvdw are the cutoffs in nm 0 9 nm 9 0 angstroms for the electrostatic and van der Waals terms The temperature coupling section is very important and must be filled out correctly Tcoupl berendsen 7 type of temperature coupling another option is nose hoover tau t Time constant for temperature coupling unit
12. 07 7 MaincChain 4H 477 8 SideChain 288 9 SideChain H 252 10 Prot Masses 765 11 Non Protein 6600 12 DRG 21 13 SOL 6579 14 Other 6600 nr group l name nr a atom amp del nr t atom type keep nr r residue res nr name group case PPPppp com com com com com com com com com com name ANnNNAHANAANAUAN DN splitch nr splitres Splitat nr chain char case sensitive nr Enter list groups l list residues h help q save and quit Use the r command to enter the list of residue numbers that represent the N amp C termini of the triple helix gt xr 1 36 37 72 73 108 15 n 1 30 37 72 3 108 51 atoms The default name r 1 36 37 72 73 108 giving to the new index group that you have just Lets rename it using the name command We will use the index group f created is cumbersome 15 in the command name 15 Terminal Turned verbose on coo 10014 CO N02 S C H pBbmpbmpbwuiwnnu O1 4 0 I9 I2 O 0 System Protein Protein H C alpha Backbone MainChain MainChain Cb MainChain H SideChain SideChain H Prot Masses Non Protein DRG SOL Other Terminal 7365 765 687 108 324 435 507 477 288 252 765 6600 21 6579 6600 Sa vooononoovownowownaoawawaa Dp com com com com com com com com com com com com com com com com ANnNDANNAANANAANANAANHNANAAUnNAAAN YN NH 14 GROMACS Tutorial nr
13. GROMACS Tutorial GROMACS Introductory Tutorial Gromacs ver 3 3 1 Author John E Kerrigan Ph D AST IST University of Medicine and Dentistry of NJ 675 Hoes Lane Piscataway NJ 08554 Phone 732 235 4473 Fax 732 235 5252 Email kerrigje umdnj edu GROMAGS Tutorial GROMAGS Tutorial for Solvation Studv of Spider Toxin Peptide Yu H Rosen M K Saccomano N A Phillips D Volkmann R A Schreiber S L Sequential assignment and structure determination of spider toxin omega Aga IVB Biochemistrv 32 pp 13123 1993 GROMAGS is a high end high performance research tool designed for the study of protein dynamics using classical molecular dynamics theory 1 This versatile software package is Gnu public license free software You may download it from http www gromacs org GROMACS runs on linux unix and on Windows a recent development Synopsis In this tutorial you will study a toxin isolated from the venom of the funnel web spider Venom toxins have been used in the past to identify cation channels Calcium ion channels regulate the entry of this ion into cells Nerve signals are highly governed by the balance of ions in neuronal cells It is hypothesized that exposed positively charged residues in venoms like the spider toxin here bind favorably to the negatively charged entrance of the cell s ion channel The spider toxin in this tutorial has its positively charged residues pointing predominantly to one side of
14. Use the which command to locate i e which cpp Content of em mdp title FWS cpp usr bin cpp the c pre processor define DFLEXIBLE constraints none integrator steep dt 0 002 ps nsteps 400 nstlist 5 ns type grid rlist 0 9 coulombtype PME rcoulomb 0 9 rvdw 1 4 fourierspacing 0 12 fourier nx 0 fourier ny 0 fourier nz 0 pme order 4 ewald rtol le 5 optimize fft yes E Energy minimizing stuff GROMAGS Tutorial emtol emstep 1000 0 0 01 Important aspects of the em mdp file title The title can be any given text description limit 64 characters keep it short and simple cpp location of the pre processor define defines to pass to the pre processor DFLEXIBLE will tell grompp to include the flexible SPC water model instead of the rigid SPC into your topology This allows steepest descents to minimize further constraints sets any constraints used in the model integrator steep tells grompp that this run is a steepest descents minimization Use cg for conjugate gradient dt not necessary for minimization Only needed for dynamics integrators like md nsteps In minimization runs this is just the maximum number of iterations nstlist frequency to update the neighbor list nstlist 10 updates every 10 steps rlist cut off distance for short range neighbor list coulombtype
15. e chains However beware as Deep View will mark those rebuilt side chains with a strange control character that can only be GROMAGS Tutorial removed manually using a text editor There are no missing side chains in this pdb file so we will not worry about that in this exercise Open 1OMB PDB in Deep View type spdbv 1OMB pdb in your unix shell Deep View fixes the structure and replaces a missing oxygen atom in a proline residue Use File gt Save gt Layer and save the new file as fws pdb Edit the file in any text editor and make sure that the HEADER and COMPND line have a name actually any name will do In addition delete the SPDBV lines added to the end of the file by DeepView HEADER FWS TOXIN COMPND FWS TOXIN Create a subdirectory in your unix account called fwspider Within this new directory create the following directories invacuo wet and ionwet Use sftp to copy your fws pdb file to the fwspider subdirectories place a copy in each subdirectory within the fwspider directory IMPORTANT Whenever you sftp a text file to a unix system from Windows be sure to convert the file to a unix text file Do this using the to_unix command e g to_unix filename filename converts filename to a unix text file In RedHat Linux use the dos2unix command Text editors in Windows like MS Word add control characters that may cause errors in unix programs Process the pdb file with pdb2gmx The pdb2gmx to v
16. iew the command line options for this command just type pdb2gmx h In fact to get help for any command in Gromacs just use the h flag command converts your pdb file to a gromacs file and writes the topology for you This file is derived from an NMR structure which contains hydrogen atoms Therefore we use the ignh flag to ignore hydrogen atoms in the pdb file The ff flag is used to select the forcefield G43al is the Gromos 96 FF a united atom FF The f flag reads the pdb file The o flag outputs a new pdb file various file formats supported with the name you have given it in the command line The p flag is used for output and naming of the topology file The topology file is very important as it contains all of the forcefield parameters based upon the forcefield that you select in the initial prompt for your model pdb2gmx ignh ff G43al f 1OMB pdb o fws pdb p fws top Setup the Box for vour simulations editconf bt cubic f fws pdb o fws pdb c d 0 9 What vou have done in this command is specifv that vou want to use a simple cubic periodic box The d 0 9 flag sets the dimensions of the box based upon setting the box edge approx 0 9 nm i e 9 0 angstroms from the molecule s peripherv We are using a small dimension for convenience and speed The molecule is centered in the box by the c flag Ideally you should set d at no less than 0 85 nm for most systems 2 GROMACS Tutorial Specia
17. ight mouse button to zoom out To view another group in the model go to Display gt Filter and the initial dialog box will come up again and you may opt to view some other index group e g the backbone To view the animation of the MD trajectory go to Display gt Animate Controls used for the playback of the trajectory appear at the bottom of the window Use the center arrow button to view one time step at time Use the forward double arrow to play the whole trajectory and use the pause button to the right to stop the trajectory playback Use the double arrow to the left to reset the animation Unfortunately the save as pdb option under the File menu in ngmx does not work Therefore 12 GROMAGS Tutorial The best wav to view the trajectorv and select snapshots to save as PDB files is to use visual molecular dynamics VMD Download from http www ks uiuc edu Research vmd It s free to academics and it runs on unix and windows Analysis One of the major advantages of Gromacs other than the fact that it is GNU public domain and free is the robust set of programs available for analyzing the trajectories We will discuss a few of the most important analytic tools in the Gromacs arsenal here Groups make_ndx The make_ndx program is useful for generating groups ID tags for specific atoms or residues that you may want to analyze Gromacs generates default groups which may be adequate for your work as is
18. l Note editconf may also be used to convert gromacs files gro to pdb files pdb and vice versa For example editconf f f le gro o file pdb converts file gro to the pdb file file pdb You may use the files generated from the this step to begin your in vacuo simulation For the in vacuo work just move ahead to the energy minimization step followed by the molecular dynamics step No position restrained dynamics necessary for in vacuo work Why Solvate the Box genbox cp fws pdb cs spc216 gro o fws_b4em pdb p fws top The genbox command generates the water box based upon the dimensions box type that you had specified using editconf In the command above you are using the SPC water model 3 The genbox program will add the correct number of water molecules needed to solvate your box of given dimensions Setup the energy minimization Use the em mdp file Gromacs uses special mdp files to setup the parameters for every type of calculation that it performs Look into the contents of this file It specifies a steepest descents minimization to remove bad van der Waals contacts Edit the file and change nsteps to 400 If the minimization fails to converge re submit with nsteps 500 The minimization should converge in less than 400 steps however different platforms respond differently To re submit the job you will need to re run grompp Note the path to the c pre processor may be different on your machine
19. o no Setting da to no instructs g hbond to analyze the rpa distance See the Gromacs manual for more options g saltbr Use g saltbr to analvze salt bridges between residues in vour simulation The program outputs a set of xvg files which give distances between minus minus charged residues plus minus the most interesting and plus plus charged residues g saltbr f fws md trr s fws md tpr How To Save specific time points from a trajectory as PDB files To get a specific frame 3000 ps in this example instead of the whole trajectory as a pdb file use additionally the dump option e g trjconv f traj xtc s file tpr o time 3000ps pdb dump 3000 20 GROMACS Tutorial Results All sample data was collected using Gomacs version 3 3 1 in single precision Potential Energy Plot Gromacs Energies 171e405 T T Potential 1 715e405 m 1 72e 05 1 725e405 1 735e405 1 74e405 1 1 TS 50 100 Time ps Computation of structure average First compute the average structure from each dynamics trajectory using either g covar or g rmsf In a real research run you would not necessarily compute the average of the entire trajectory You would just compute the average for the time points where you have reached some equilibrium In our run we computed the average for 60 ps of simulation using g rmsf it s faster g rmsf ffws md trr s fws md tpr b 30 e 90
20. odicity on Peptide Conformation J Phys Chem B 2000 104 15 p 3668 3575 3 Berendsen H J J P Postma W F van Gunsteren and J Hermans Interaction models for water in relation to protein hydration in Intermolecular Forces B Pullman Editor 1981 D Reidel Publishing Co Dordrecht p 331 342 4 Essmann U L Perera M L Berkowitz T Darden H Lee and L Pedersen A smooth particle mesh ewald potential J Chem Phys 1995 103 p 8577 8592 5 Darden T D York and L Pedersen Particle Mesh Ewald An N log N method for Ewald sums in large systems J Chem Phys 1993 98 p 10089 10092 6 Hess B H Bekker H Berendsen and J Fraaije LINCS A Linear Constraint Solver for molecular simulations J Comp Chem 1997 18 p 1463 1472 7 Berendsen H J C J P M Postma W F vanGunsteren A DiNola and J R Haak Molecular dynamics with coupling to an external bath J Chem Phys 1984 81 8 p 3584 3590 22
21. rfTen Pcoupl Mu XX Pcoupl Mu YY Pcoupl Mu ZZ Mu X Mu Y Mu Z T Protein T SOL T CL Lamb Protein Lamb SOL Lamb CL For potential energy type Potential lt enter gt Hit the lt enter gt key again We get a summary with average PE and RMSD in kJ mol Last frame read 500 time 100 000 Statistics over 50001 steps 0 0000 thru 100 0000 ps 1 data sets Energy Average RMSD Fliet Drift Tot Drift Potential 172216 287 932 287 925 0 0672381 6 72394 To read xvg files into Grace use the following command xmgrace nxy fws pe xvg Grace is not available on UMDNJ computers at this time You may obtain Grace from http plasma gate weizmann ac il Grace Grace runs on Linux and in Unix only Ifyou do not have Grace or Xmgr just import or read the xvg file into MS Excel as a space delimited file g gyrate 16 GROMAGS Tutorial Use g gvrate to measure the radius of gyration This quantity gives a measure of the compactness of the structure This gives a measure of the mass of the atom s relative the center of mass of the molecule g gyrate f fws md trr s fws md tpr o fws gyrate xvg g rms and g rmsdist These programs are useful for measuring root mean square deviations in the structure Use g rms to evaluate the deviation of the structure from the original starting structure over the course of the simulation The dt 10 option tells the program to write every tenth frame g rms s tpr f xtc dt 1
22. s ps You must list one per tc_grp in the order in which tc grps appear tc grps groups to couple to the thermostat every atom or residue in your model must be represented here by appropriate index groups ref t reference temperature for coupling i e the temperature of your MD simulation in degrees K You must list one per tc grp in the order in which tc grps appear When you alter the temperature don t forget to change the gen temp variable for velocity generation pcoupl is the berendsen barostat for controlling the simulation pressure 7 GROMAGS Tutorial pcoupltype isotropic means that the box will expand or contract evenly in all directions x y and z in order to maintain the proper pressure tau p time constant for coupling in ps compressibility this is the compressibility of the solvent used in the simulation in bar the setting above is for water at 300 K and 1 atm ref p the reference pressure for the coupling in bar 1 atm 0 983 bar Pre process the pr mdp file grompp f pr mdp c fws b4pr pdb r fws b4pr pdb p fws top o fws pr tpr mdrun s fws pr tpr o fws pr trr c fws b4md pdb g pr log e pr edr amp Use the tail command to check the pr log file The md mdp parameter file looks very similar to the pr mdp file There are several differences The define statement is not necessary as we are not running as position restrained dynamics Conten
23. t of md mdp for explicit solvation Special Note For in vacuo remove the entries for sol in the temperature coupling section For the simulation that includes the counterions add an entry for the ions in the temperature coupling section title FWS cpp usr bin cpp constraints all bonds integrator md dt 0 002 ps nsteps 25000 total 50 ps nstcomm Ex od nstxout 500 collect data every 1 ps nstvout 0 nstfout 0 nstlist 5 ns type grid rlist 0 9 coulombtype PME rcoulomb 0 9 rvdw 1 4 fourierspacing 0 12 fourier nx 0 fourier ny 0 fourier nz 0 pme_order 4 ewald rtol le 5 optimize fft yes Berendsen temperature coupling is on in two groups Tcoupl berendsen tau_t 0 1 0 1 0 1 tc grps protein sol CL ref t 300 300 300 GROMAGS Tutorial Pressure coupling is on Pcoupl berendsen tau p 0 5 compressibility 4 5e 5 ref p 1 0 Generate velocites is on at 300 K gen vel yes gen temp 300 0 gen seed 173529 grompp f md mdp c fws b4md pdb r fws_b4md pdb p fws top o fws md tpr mdrun s fws md tpr o fws md trr c fws pmd pdb g md log e md edr amp Use the tail command to check the md log file You may compress the trajectory using trjconv to save on disk space trjconv f filename trr o filename xtc Once you have made the xtc file you may delete the trr file Use ngmx
24. tells gromacs how to model electrostatics PME is particle mesh ewald method please see the Gromacs user manual for more information rcoulomb distance for the coulomb cut off rvdw distance for the LJ or Buckingham potential cut off EM Stuff emtol the minimization converges when the max force is smaller than this value in units of kJ mol nm emstep initial step size in nm Now process the files with grompp grompp is the pre processor program the gromacs pre processor grompp Getitl Sigh grompp will setup your run for input into mdrun grompp f em mdp c fws b4em pdb p fws top o fws em tpr The f flag in grompp is used to input the parameter file mdp The c flag is used to input the coordinate file the pdb file pdb p inputs the topology and o outputs the input file tpr needed for mdrun Using genion and the tpr file to add ions You may use the tpr file generated here to add counterions to your model to neutralize any net charge Our model has a net charge of 2 00 Therefore we want to add two chloride ions Copy the fws em tpr file that you used for your explicit solvated model to your ionwet subdirectory In addition copy your fws top and posre itp files from your explicit solvation model to your ionwet subdirectory Use the genion command to add the chloride ions genion s fws em tpr o fws ion pdb nname CL nn 2 g fws ion log GROMACS Tutorial
25. the peptide The ion channel is blocked resulting in blocked nerve signal leading to paralysis and ultimately to death presumably via respiratory failure We will study this peptide toxin using explicit solvation dynamics First we will compare an in vacuo model to a solvated model We will solvate the peptide in a water box followed by equilibration using Newton s laws of motion We will compare and contrast the impact of counterions in the explicit solvation simulation We will seek answers to the following questions Is the secondary structure stable to the dynamics conditions Are the side chains of the positively charged residues predominantly displaced to one side of the peptide structure Do the counterions hold these positively charged residues in place or do they move around What role does water play in maintaining the structure of proteins Note You will generate gromacs gro structure files in this tutorial To view these files you must use VMD Download from http www ks uiuc edu Research vmd In addition you should obtain a copy of the GROMACS user manual at http www gromacs org Download 1OMB PDB from the Protein Data Bank http www resb org pdb We will use MOE to preview the PDB file It is advisable to use DeepView Download DeepView from http www expasy ch spdbv if you know that your structure may be disordered i e residues with missing side chains Deep View will replace any missing sid
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