All instructions require prior GROMACS installation. All simulations for this manuscript were run using the Holland Computing Center with GROMACS installed.
## Getting started
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.
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.
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.
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
Analyzing MD Simulations
-[ ] [Create](https://docs.gitlab.com/ee/user/project/repository/web_editor.html#create-a-file) or [upload](https://docs.gitlab.com/ee/user/project/repository/web_editor.html#upload-a-file) files
-[ ] [Add files using the command line](https://docs.gitlab.com/ee/gitlab-basics/add-file.html#add-a-file-using-the-command-line) or push an existing Git repository with the following command:
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.
## Integrate with your tools
RMSD
-[ ] [Set up project integrations](https://git.unl.edu/powers-group/2022-biopolymers-supplemental/-/settings/integrations)
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.
## Collaborate with your team
Radius of Gyration
-[ ] [Invite team members and collaborators](https://docs.gitlab.com/ee/user/project/members/)
-[ ] [Create a new merge request](https://docs.gitlab.com/ee/user/project/merge_requests/creating_merge_requests.html)
-[ ] [Automatically close issues from merge requests](https://docs.gitlab.com/ee/user/project/issues/managing_issues.html#closing-issues-automatically)
-[ ] [Automatically merge when pipeline succeeds](https://docs.gitlab.com/ee/user/project/merge_requests/merge_when_pipeline_succeeds.html)
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.
## Test and Deploy
SASA
Use the built-in continuous integration in GitLab.
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.
-[ ] [Get started with GitLab CI/CD](https://docs.gitlab.com/ee/ci/quick_start/index.html)
-[ ] [Analyze your code for known vulnerabilities with Static Application Security Testing(SAST)](https://docs.gitlab.com/ee/user/application_security/sast/)
-[ ] [Deploy to Kubernetes, Amazon EC2, or Amazon ECS using Auto Deploy](https://docs.gitlab.com/ee/topics/autodevops/requirements.html)
-[ ] [Use pull-based deployments for improved Kubernetes management](https://docs.gitlab.com/ee/user/clusters/agent/)
-[ ] [Set up protected environments](https://docs.gitlab.com/ee/ci/environments/protected_environments.html)
***
# Editing this README
When you're ready to make this README your own, just edit this file and use the handy template below (or feel free to structure it however you want - this is just a starting point!). Thank you to [makeareadme.com](https://www.makeareadme.com/) for this template.
iRed Analysis of MD Simulations
## Suggestions for a good README
Every project is different, so consider which of these sections apply to yours. The sections used in the template are suggestions for most open source projects. Also keep in mind that while a README can be too long and detailed, too long is better than too short. If you think your README is too long, consider utilizing another form of documentation rather than cutting out information.
The analysis utilizes iRed software provided by the Dr. Rafael Bruschweiler Group. All iRed calculations were completed on the Holland Computing Center cluster.
## Name
Choose a self-explaining name for your project.
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.
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.
## Description
Let people know what your project can do specifically. Provide context and add a link to any reference visitors might be unfamiliar with. A list of Features or a Background subsection can also be added here. If there are alternatives to your project, this is a good place to list differentiating factors.
## Badges
On some READMEs, you may see small images that convey metadata, such as whether or not all the tests are passing for the project. You can use Shields to add some to your README. Many services also have instructions for adding a badge.
## Visuals
Depending on what you are making, it can be a good idea to include screenshots or even a video (you'll frequently see GIFs rather than actual videos). Tools like ttygif can help, but check out Asciinema for a more sophisticated method.
## Installation
Within a particular ecosystem, there may be a common way of installing things, such as using Yarn, NuGet, or Homebrew. However, consider the possibility that whoever is reading your README is a novice and would like more guidance. Listing specific steps helps remove ambiguity and gets people to using your project as quickly as possible. If it only runs in a specific context like a particular programming language version or operating system or has dependencies that have to be installed manually, also add a Requirements subsection.
## Usage
Use examples liberally, and show the expected output if you can. It's helpful to have inline the smallest example of usage that you can demonstrate, while providing links to more sophisticated examples if they are too long to reasonably include in the README.
## Support
Tell people where they can go to for help. It can be any combination of an issue tracker, a chat room, an email address, etc.
## Roadmap
If you have ideas for releases in the future, it is a good idea to list them in the README.
## Contributing
State if you are open to contributions and what your requirements are for accepting them.
For people who want to make changes to your project, it's helpful to have some documentation on how to get started. Perhaps there is a script that they should run or some environment variables that they need to set. Make these steps explicit. These instructions could also be useful to your future self.
You can also document commands to lint the code or run tests. These steps help to ensure high code quality and reduce the likelihood that the changes inadvertently break something. Having instructions for running tests is especially helpful if it requires external setup, such as starting a Selenium server for testing in a browser.
## Authors and acknowledgment
Show your appreciation to those who have contributed to the project.
## License
For open source projects, say how it is licensed.
## Project status
If you have run out of energy or time for your project, put a note at the top of the README saying that development has slowed down or stopped completely. Someone may choose to fork your project or volunteer to step in as a maintainer or owner, allowing your project to keep going. You can also make an explicit request for maintainers.
