diff --git a/README.md b/README.md
index b693e6665d73b6228dcd662f5ab6e94445e2c7c2..77dc6ca507a079b3be1039c1c632b5ff1ed90523 100644
--- a/README.md
+++ b/README.md
@@ -1,92 +1,77 @@
# 2022-biopolymers-supplemental
-
-
-## Getting started
-
-To make it easy for you to get started with GitLab, here's a list of recommended next steps.
-
-Already a pro? Just edit this README.md and make it your own. Want to make it easy? [Use the template at the bottom](#editing-this-readme)!
-
-## Add your files
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-
-```
-cd existing_repo
-git remote add origin https://git.unl.edu/powers-group/2022-biopolymers-supplemental.git
-git branch -M main
-git push -uf origin main
-```
-
-## Integrate with your tools
-
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-## Collaborate with your team
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-
-## Test and Deploy
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-
-***
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-# Editing this README
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+Setting up and Running an MD Simulation
+
+All instructions require prior GROMACS installation. All simulations for this manuscript were run using the Holland Computing Center with GROMACS installed.
+
+1. Download required PDB file from https://www.rcsb.org. In this case, 2OR3.
+2. Move downloaded PDB file to desired folder location where MD simulations will be run and rename 2or3.pdb.
+3. Using the command line, navigate to the working folder where PDB file is located.
+4. Use command "grep -v HOH 2or3.pdb > 2or3_clean.pdb” to remove water from crystal structures.
+1. If PDB structure has other atoms, such as SO4, ligands or PO4, run this command again, replacing HOH with other atom.
+2. This can also be accomplished by manually removing the HOH, SO4, PO4, etc. lines in the PDB file using a text editor.
+5. Use command "gmx pdb2gmx -f 2or3_clean.pdb -o 2or3_processed.gro -water spce”.
+1. This step will prompt you to chose a force field. A commonly used field is AMBER99SB and can be selected by typing “5” and pressing enter. This step converts the pdb file into a GROMACS file to be used in subsequent steps.
+6. Solvate the system by running "gmx editconf -f 2or3_processed.gro -o 2or3_newbox.gro -c -d 1.0 -bt cubic" and "gmx solvate -cp 2or3_newbox.gro -cs spc216.gro -o 2or3_solv.gro -p topol.top”
+1. These commands create a 1 nm box around the protein and fills that box with water molecules.
+7. The next steps require a series of .mdp files. These files are all found here. These files should be added to the working folder.
+8. Run the commands "gmx grompp -f ions.mdp -c 2or3_solv.gro -p topol.top -o ions.tpr" and "gmx genion -s ions.tpr -o 2or3_solv_ions.gro -p topol.top -pname NA -nname CL -neutral”
+1. To run at physiological salt conditions, add the "-conc 0.1" flag before -pname.
+2. This adds the necessary ions to the solvent within the 1 nm box.
+9. Once solvent and ions are added, the system is ready for minimization. Edit "minim.mdp" to the desired number of steps (50,000 is fairly standard for this step). Run "gmx grompp -f minim.mdp -c 2or3_solv_ions.gro -p topol.top -o em.tpr" and gmx mdrun -v -deffnm em".
+1. Once minimization is complete, run command "gmx energy -f em.edr -o potential.xvg", inputting "10 0" when prompted and open .xvg file to check plot. Plot should show steady convergence of Epot.
+10. The next step is to equilibrate the temperature of the system. Edit the nvt.mdp file to run for 100ps by changing nsteps to 50,000. The reference temperature you want to run the simulation at can also be edited here by changing ref_t.
+1. Run commands "gmx grompp -f nvt.mdp -c em.gro -r em.gro -p topol.top -o nvt.tpr" and "gmx mdrun -deffnm nvt”.
+2. Plot these results using "gmx energy -f nvt.edr -o temperature.xvg" inputting "16 0" when prompted
+3. If temperature doesn't remain stable at the target temperature, a longer equilibration is necessary.
+11. Next, equilibrate the density of the system. Edit the npt.mdp as you did the nvt.mdp.
+1. Run commands "gmx grompp -f npt.mdp -c nvt.gro -r nvt.gro -t nvt.cpt -p topol.top -o npt.tpr" and "gmx mdrun -deffnm npt"
+2. Plot results using "gmx energy -f npt.edr -o pressure.xvg" inputting "18 0" when prompted.
+12. The system is now ready for the production simulation. Edit the md.mdp to run for the desired length (500,000 steps is a 1ns simulation). Edit the reference temperature to desired temperature.
+1. Run commands "gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -o md_0_1.tpr" and "gmx mdrun -deffnm md_0_1”
+2. Note: longer simulations may require the use of GPU cores.
+
+
+
+Analyzing MD Simulations
+
+All analysis of MD simulations were done using the Holland Computing Center.
+
+1. Once the simulation is done running, the first step is to convert the trajectory. Do this by running “gmx trjconv -s md_0_1.tpr -f md_0_1.xtc -o md_0_1_noPBC.xtc -pbc mol -center”.
+1. The -pbc flag corrects for periodicity and may need to be changed based on specific protein simulations. Instructions for changing this flag can be found in the GROMACS manual (https://manual.gromacs.org/2020/manual-2020.pdf).
+2. Once the .xtc file is created, many different analyses can be completed using the same general commands. The GROMACS manual is the best place to find useful commands, as well as a list of analyses that can be completed.
+1. Several of the most common analyses are RMSD, radius of gyration and solvent accessible surface area (SASA). The commands for these analyses are below.
+
+RMSD
+
+1. Run the command “gmx rms -s md_0_1.tpr -f md_0_1_noPBC.xtc -o rmsd.xvg -tu ns”, making sure that input files -s and -f match exactly the naming conventions you used previously.
+1. Input “4” and “4” when prompted.
+2. Outputs RMSD values in ns for the duration of the simulation.
+
+Radius of Gyration
+
+1. Run the command “gmx gyrate -s md_0_1.tpr -f md_0_1_noPBC.xtc -o gyrate.xvg”. Input “1” when prompted.
+1. Outputs radius of gyration values in ps for the duration of the simulation.
+
+SASA
+
+1. Run the command “gmx sasa -s md_0_1.tpr -f traj1.xtc -tu ns -o sasa.xvg”, inputing “1” when prompted.
+1. Outputs SASA values in ns for the duration of the simulation.
+
+
+
+
+iRed Analysis of MD Simulations
+
+The analysis utilizes iRed software provided by the Dr. Rafael Bruschweiler Group. All iRed calculations were completed on the Holland Computing Center cluster.
+
+1. Create a pdb file of the trajectory using “gmx trjconv -s md_0_1.tpr -f md_0_1.xtc -o md_0_1_noPBC.pdb -pbc mol -center”
+1. The generated pdb file will be very large and the conversion may take some time.
+2. Use the following commands to extract N and H coordinates for each atom at each time point in the trajectory.
+1. awk ‘{if (($3 == “N”) && ($4 != “PRO”)) {print $6, $7, $8, $9:}} xxx.pdb > N-HCoor.dat
+2. awk ‘{if (($3 == “H”) && ($4 != “H1”)) {print $6, $7, $8, $9:}} xxx.pdb > H-NCoor.dat
+3. Once .dat files are created, use the python script below to run the iRed calculation
+1. In command line, run “ired_1vec_block.py --coor1 N-HCoor.dat --coor2 H-NCoor.dat --block 100”
+2. Python script will output a .out file that contains the order parameter values for each amino acid residue.
+
+
diff --git a/ions.mdp b/ions.mdp
new file mode 100644
index 0000000000000000000000000000000000000000..7348b9efef562137f7615436b8d72d2b6b9f86de
--- /dev/null
+++ b/ions.mdp
@@ -0,0 +1,15 @@
+; ions.mdp - used as input into grompp to generate ions.tpr
+; Parameters describing what to do, when to stop and what to save
+integrator = steep ; Algorithm (steep = steepest descent minimization)
+emtol = 1000.0 ; Stop minimization when the maximum force < 1000.0 kJ/mol/nm
+emstep = 0.01 ; Minimization step size
+nsteps = 50000 ; Maximum number of (minimization) steps to perform
+
+; Parameters describing how to find the neighbors of each atom and how to calculate the interactions
+nstlist = 1 ; Frequency to update the neighbor list and long range forces
+cutoff-scheme = Verlet ; Buffered neighbor searching
+ns_type = grid ; Method to determine neighbor list (simple, grid)
+coulombtype = cutoff ; Treatment of long range electrostatic interactions
+rcoulomb = 1.0 ; Short-range electrostatic cut-off
+rvdw = 1.0 ; Short-range Van der Waals cut-off
+pbc = xyz ; Periodic Boundary Conditions in all 3 dimensions
\ No newline at end of file
diff --git a/md.mdp b/md.mdp
new file mode 100644
index 0000000000000000000000000000000000000000..82378160d8236c14177af83d4a0ef69396eccd6d
--- /dev/null
+++ b/md.mdp
@@ -0,0 +1,46 @@
+title = OPLS Lysozyme NPT equilibration
+; Run parameters
+integrator = md ; leap-frog integrator
+nsteps = 500000 ; 2 * 500000 = 1000 ps (1 ns)
+dt = 0.002 ; 2 fs
+; Output control
+nstxout = 0 ; suppress bulky .trr file by specifying
+nstvout = 0 ; 0 for output frequency of nstxout,
+nstfout = 0 ; nstvout, and nstfout
+nstenergy = 5000 ; save energies every 10.0 ps
+nstlog = 5000 ; update log file every 10.0 ps
+nstxout-compressed = 5000 ; save compressed coordinates every 10.0 ps
+compressed-x-grps = System ; save the whole system
+; Bond parameters
+continuation = yes ; Restarting after NPT
+constraint_algorithm = lincs ; holonomic constraints
+constraints = h-bonds ; bonds involving H are constrained
+lincs_iter = 1 ; accuracy of LINCS
+lincs_order = 4 ; also related to accuracy
+; Neighborsearching
+cutoff-scheme = Verlet ; Buffered neighbor searching
+ns_type = grid ; search neighboring grid cells
+nstlist = 10 ; 20 fs, largely irrelevant with Verlet scheme
+rcoulomb = 1.0 ; short-range electrostatic cutoff (in nm)
+rvdw = 1.0 ; short-range van der Waals cutoff (in nm)
+; Electrostatics
+coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics
+pme_order = 4 ; cubic interpolation
+fourierspacing = 0.16 ; grid spacing for FFT
+; Temperature coupling is on
+tcoupl = V-rescale ; modified Berendsen thermostat
+tc-grps = Protein Non-Protein ; two coupling groups - more accurate
+tau_t = 0.1 0.1 ; time constant, in ps
+ref_t = 300 300 ; reference temperature, one for each group, in K
+; Pressure coupling is on
+pcoupl = Parrinello-Rahman ; Pressure coupling on in NPT
+pcoupltype = isotropic ; uniform scaling of box vectors
+tau_p = 2.0 ; time constant, in ps
+ref_p = 1.0 ; reference pressure, in bar
+compressibility = 4.5e-5 ; isothermal compressibility of water, bar^-1
+; Periodic boundary conditions
+pbc = xyz ; 3-D PBC
+; Dispersion correction
+DispCorr = EnerPres ; account for cut-off vdW scheme
+; Velocity generation
+gen_vel = no ; Velocity generation is off
\ No newline at end of file
diff --git a/minim.mdp b/minim.mdp
new file mode 100644
index 0000000000000000000000000000000000000000..7ccebada3da8be3885305da4e96cbc292b933a0e
--- /dev/null
+++ b/minim.mdp
@@ -0,0 +1,15 @@
+; minim.mdp - used as input into grompp to generate em.tpr
+; Parameters describing what to do, when to stop and what to save
+integrator = steep ; Algorithm (steep = steepest descent minimization)
+emtol = 1000.0 ; Stop minimization when the maximum force < 1000.0 kJ/mol/nm
+emstep = 0.01 ; Minimization step size
+nsteps = 50000 ; Maximum number of (minimization) steps to perform
+
+; Parameters describing how to find the neighbors of each atom and how to calculate the interactions
+nstlist = 1 ; Frequency to update the neighbor list and long range forces
+cutoff-scheme = Verlet ; Buffered neighbor searching
+ns_type = grid ; Method to determine neighbor list (simple, grid)
+coulombtype = PME ; Treatment of long range electrostatic interactions
+rcoulomb = 1.0 ; Short-range electrostatic cut-off
+rvdw = 1.0 ; Short-range Van der Waals cut-off
+pbc = xyz ; Periodic Boundary Conditions in all 3 dimensions
\ No newline at end of file
diff --git a/npt.mdp b/npt.mdp
new file mode 100644
index 0000000000000000000000000000000000000000..a5e868da96b768a2548c76bfea0ebc8d92456cc4
--- /dev/null
+++ b/npt.mdp
@@ -0,0 +1,44 @@
+title = OPLS Lysozyme NPT equilibration
+define = -DPOSRES ; position restrain the protein
+; Run parameters
+integrator = md ; leap-frog integrator
+nsteps = 50000 ; 2 * 50000 = 100 ps
+dt = 0.002 ; 2 fs
+; Output control
+nstxout = 500 ; save coordinates every 1.0 ps
+nstvout = 500 ; save velocities every 1.0 ps
+nstenergy = 500 ; save energies every 1.0 ps
+nstlog = 500 ; update log file every 1.0 ps
+; Bond parameters
+continuation = yes ; Restarting after NVT
+constraint_algorithm = lincs ; holonomic constraints
+constraints = h-bonds ; bonds involving H are constrained
+lincs_iter = 1 ; accuracy of LINCS
+lincs_order = 4 ; also related to accuracy
+; Nonbonded settings
+cutoff-scheme = Verlet ; Buffered neighbor searching
+ns_type = grid ; search neighboring grid cells
+nstlist = 10 ; 20 fs, largely irrelevant with Verlet scheme
+rcoulomb = 1.0 ; short-range electrostatic cutoff (in nm)
+rvdw = 1.0 ; short-range van der Waals cutoff (in nm)
+DispCorr = EnerPres ; account for cut-off vdW scheme
+; Electrostatics
+coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics
+pme_order = 4 ; cubic interpolation
+fourierspacing = 0.16 ; grid spacing for FFT
+; Temperature coupling is on
+tcoupl = V-rescale ; modified Berendsen thermostat
+tc-grps = Protein Non-Protein ; two coupling groups - more accurate
+tau_t = 0.1 0.1 ; time constant, in ps
+ref_t = 300 300 ; reference temperature, one for each group, in K
+; Pressure coupling is on
+pcoupl = Parrinello-Rahman ; Pressure coupling on in NPT
+pcoupltype = isotropic ; uniform scaling of box vectors
+tau_p = 2.0 ; time constant, in ps
+ref_p = 1.0 ; reference pressure, in bar
+compressibility = 4.5e-5 ; isothermal compressibility of water, bar^-1
+refcoord_scaling = com
+; Periodic boundary conditions
+pbc = xyz ; 3-D PBC
+; Velocity generation
+gen_vel = no ; Velocity generation is off
\ No newline at end of file
diff --git a/nvt.mdp b/nvt.mdp
new file mode 100644
index 0000000000000000000000000000000000000000..a0682d6c9b78898568815380af519818588c8138
--- /dev/null
+++ b/nvt.mdp
@@ -0,0 +1,41 @@
+title = OPLS Lysozyme NVT equilibration
+define = -DPOSRES ; position restrain the protein
+; Run parameters
+integrator = md ; leap-frog integrator
+nsteps = 50000 ; 2 * 50000 = 100 ps
+dt = 0.002 ; 2 fs
+; Output control
+nstxout = 500 ; save coordinates every 1.0 ps
+nstvout = 500 ; save velocities every 1.0 ps
+nstenergy = 500 ; save energies every 1.0 ps
+nstlog = 500 ; update log file every 1.0 ps
+; Bond parameters
+continuation = no ; first dynamics run
+constraint_algorithm = lincs ; holonomic constraints
+constraints = h-bonds ; bonds involving H are constrained
+lincs_iter = 1 ; accuracy of LINCS
+lincs_order = 4 ; also related to accuracy
+; Nonbonded settings
+cutoff-scheme = Verlet ; Buffered neighbor searching
+ns_type = grid ; search neighboring grid cells
+nstlist = 10 ; 20 fs, largely irrelevant with Verlet
+rcoulomb = 1.0 ; short-range electrostatic cutoff (in nm)
+rvdw = 1.0 ; short-range van der Waals cutoff (in nm)
+DispCorr = EnerPres ; account for cut-off vdW scheme
+; Electrostatics
+coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics
+pme_order = 4 ; cubic interpolation
+fourierspacing = 0.16 ; grid spacing for FFT
+; Temperature coupling is on
+tcoupl = V-rescale ; modified Berendsen thermostat
+tc-grps = Protein Non-Protein ; two coupling groups - more accurate
+tau_t = 0.1 0.1 ; time constant, in ps
+ref_t = 300 300 ; reference temperature, one for each group, in K
+; Pressure coupling is off
+pcoupl = no ; no pressure coupling in NVT
+; Periodic boundary conditions
+pbc = xyz ; 3-D PBC
+; Velocity generation
+gen_vel = yes ; assign velocities from Maxwell distribution
+gen_temp = 300 ; temperature for Maxwell distribution
+gen_seed = -1 ; generate a random seed
\ No newline at end of file