TITLE: Protein PEGylation Protocol - Full Martini Coarse Grained v2.0 Protocol (MARTINI, INSANE, Gromacs simulations)
AUTHORS: compt.biology.agutierrez, Carmen Ili Gangas

[DESCRIPTION]
The construction of coarse-grained systems from atomistic models is a key strategy in simulating molecular-level biological systems. This process involves reducing the complexity of an atomic system, represented by a large number of particles, to a coarser or simplified level, enabling the study of phenomena at larger time scales. To carry out this conversion, the tool insane.py can be employed, designed to generate topologies and initial configurations for coarse-grained systems.

Firstly, insane.py is used to translate an atomistic model into a coarse-grained format, defining the relationship between groups of atoms and simplifying interactions. This step is essential to reduce computational load and enable simulations at extended time scales.

Subsequently, GROMACS, a renowned molecular dynamics software suite, is utilized to perform molecular dynamics simulations on the newly generated coarse-grained system. This process allows for the study of the dynamics and behavior of the system under realistic conditions.

In the context of protein PEGylation, a common methodology to enhance protein stability and solubility, a basic approach is followed. PEGylation involves the covalent attachment of polyethylene glycol (PEG) to proteins, providing unique properties to improve drug efficacy and reduce immunogenicity. The basic methodology involves the appropriate selection of conjugation sites on the protein and the chemical binding of the PEG polymer, often through amide-type reactions.

In summary, the construction of coarse-grained systems, the use of tools like insane.py, and the application of molecular dynamics techniques with GROMACS are pivotal in simulating molecular systems. Additionally, protein PEGylation offers an effective strategy to enhance various biomolecular properties with significant applications in drug development.

[STEPS]
SECTION: INTRODUCTION
1. The field of improving proteins via PEGylation remains a vibrant area of research, yet a profound understanding of the interactions between polymers and proteins is still in its infancy. To shed light on these interactions and enable predictive insights, Molecular Dynamics (MD) simulations offer a powerful tool, particularly when focused on specific protein-polymer systems at the molecular level. In this protocol, we provide detailed instructions on simulating PEGylated proteins, leveraging the capabilities of the latest iteration of the Martini Coarse-Grained (CG) force field.

The Martini CG force field is renowned for its ability to efficiently represent large biomolecular systems by grouping several atoms into a single interaction site. This coarse-grained approach significantly reduces computational demands while maintaining a remarkable level of accuracy. Specifically designed for simulating complex biological processes, the Martini force field captures the essential features of biomolecules, making it an ideal choice for studying intricate protein-polymer interactions.

This protocol focuses on the utilization of Martini CG force field in Molecular Dynamics simulations, providing researchers with the means to obtain nearly atomistic information. Unlike fully atomistic simulations, MD simulations with CG allow for the exploration of complex biological systems over extended temporal and spatial scales. This feature is particularly valuable when investigating the dynamic behavior of PEGylated proteins and their interactions with surrounding environments.

By presenting this protocol, we aim to empower researchers to delve deeper into the molecular dynamics of protein-PEG interactions, fostering a more comprehensive understanding of how PEGylation influences protein behavior at the molecular level. Through the integration of Martini CG force field in this protocol, we provide a sophisticated yet accessible approach to unraveling the complexities inherent in these biologically relevant systems.

SECTION: MATERIALS
2. Skills Need it

Basics information skills needed:
- Install standardized software.
- Run short commands in terminal
- Construct short scripts.

Computational Biology Skills:
- Understand protein format pdb and gro.
- Understand atom types in all atoms and coarse grained structures.
- Understand the biophysics of atoms in a solvent.

SECTION: MATERIALS
4. Software Requirements

1º Python ≥ 3.11.5. 
The easiest way to get started with Python is by downloading the Anaconda distribution. (Lower version can function anyway, but its already tested on this one)
 
 
2º Polyply v1.0
This is the core software, with this one we create a variety of Polymer structures bounded to differences structures. For more information, please visit "Marrik Lab - Polyply v1.0", in GitHub for more information. 
 

3º Martini Coarse Grained
The Martini force field is a coarse-grain (CG) force field suited for molecular dynamics simulations of biomolecular systems. Could be either, installed or downloaded and executed as python scripts. Last version in this link to GitHub : "Martinize", for more information.  
 

4º Insane system constructor (Membrane Construction)
Usually used as lipid system constructor. Wide lipid structures and relatively easy to use, just need 1 command. In this tutorial, we're going to use it as system constructor, add water and ions molecules. Please, visit : "Insane, Lipidomics Tutorial", for more information. 
 

SECTION: MATERIALS
3. Hardware Requirements

1º Personal computer. Could be a workstation or laptop. 
2º 4 GB of ram is recommended. (As many laptops/workstation has more than that almost every computer will be fine)
SECTION: GROMACS FILE PREPARATIONS
11. FILES MODIFICATION PRIOR MOLECULAR DYNAMICS : ITP and TOPOL files.
ITP and Topol files modification. 

Prior run MD, we will create a folder called LYSOZYME_MD. In this folder we will create 2 new folders, with the following names :
ITP
MDP
And copy the files generated in Insane (system.gro & topol.top) and Martini (molecule_0.itp) steps into LYZOSYME_MD folder. This its just to have everything organized, its nor necessary but its useful. 

<Topol.top>
This file is created by insane with default filepaths, so it is necessary to correct them to match the filepath of our files. 
 

<molecule_0.itp>
This file is created by martini in step 6, but its useful modify some arguments in it to control some characteristics. 
 

Output file after modification:
  
  

Position Restraints Modification : This modification will lead us to control the Position Restraints value from MDP files. While we enter into Equilibration steps in molecular dynamics, we want to decrease the Position_Restraints gradually, until reach the lowest Position Restraints (value of your preferences) before Production Run.

RUBBER_BANDS Modification : This modification will lead us to activate Rubber bands (our elastic networks values) from MDP files. 

SECTION: GROMACS MOLECULAR DYNAMICS
15. MOLECULAR DYNAMICS GROMACS v2019 - SIMULATION
General commands and usage.

GROMACS stands out as essential software for simulating molecular dynamics (MD) in coarse-grained structures, simplifying representations and enabling efficient large-scale studies. Its balanced approach between accuracy and computational efficiency makes it crucial in biomolecular research.

To run a molecular dynamics you need to execute 2 commands per phase. We understand that we will have x amount of phases as x amount of mdp files. Therefore, two commands per phase are required :

<grompp command>
The first, the grompp command, will load data from both the mdp, itp, system.gro, index.ndx and topol.top files.

<mdrun command>
The second, mdrun command, executes the parameters loaded to Gromacs with the grompp command. In this command you specify how many cores to use, whether to use gpu and the name of the output files.

As an example, here is the command to run the energy minimization phase :
 
Finally, to pass from the energy minimization stage to NVT equilibration, the files generated previously in the mdrun command (should be minimization.gro) must be used to generate the new NVT files.
 

SECTION: GROMACS FILE PREPARATIONS
12. FILES MODIFICATION PRIOR MOLECULAR DYNAMICS :Input Parameters files.
Input parameters explanation and modification. 

Input files can be downloaded from Martini Coarse Grained official website -> Martini Coarse Grained. You can modify some values depending in what do you want and your expertise in biophysics. .

<Standard Input Parameters File Attached in section 10> 

Minimization Parameters:  The solvated, electroneutral system is now assembled. Before we can begin dynamics, we must ensure that the system has no steric clashes or inappropriate geometry. The structure is relaxed through a process called energy minimization (EM).

Equilibration Parameters: Equilibration is often conducted in two phases, NVT and NPT phases. The first phase is conducted under an NVT ensemble (constant Number of particles, Volume, and Temperature). This ensemble is also referred to as "isothermal-isochoric" or "canonical." And, equilibration of pressure is conducted under an NPT ensemble, wherein the Number of particles, Pressure, and Temperature are all constant. 
In coarse grained system, its not need it an NVT equilibration, so just perform the quantity needed NPT equilibrations modificating Position restraints forces. For this tutorial we will use an approach in which we will increase the time in the "dt" option from 1 to 15 femtoseconds, increasing 5 femtoseconds for each mdp balance file, so 4 NPT equilibration processes will be performed.
In addition to the above, the positional constraint values will gradually decrease from 1000 to 125 kJ/mol nm2, decreasing by half to 125 kJ/mol nm2.

Production Parameters: Upon completion of the two equilibration phases, the system is now well-equilibrated at the desired temperature and pressure. We are now ready to release the position restraints and run production MD for data collection. The process is just like we have seen before, as we will make use of the checkpoint file (which in this case now contains preserve pressure coupling information) to grompp. 

Almost every parameter in mdp file can be modified. If you download mdp files from martini website, unless you really know what to change in this parameters, as an advice "don't change the parameters". These defaults parameters are already tested. 

SECTION: GROMACS FILE PREPARATIONS
13. FILES GENERATION PRIOR MOLECULAR DYNAMICS : index files.
Index File explanation and generation.

The GROMACS index file (usually called index.ndx) contains some user definable sets of atoms. The file can be read by most analysis Gromacs programs. One of the most easiest files generated by Gromacs, just need 1 command. 
 
Unless you need to create a special index group, just use this short command to generate your index file. 

SECTION: MARTINIZE &amp; INSANE PROTOCOL
5. FILES USED IN THIS METHODOLOGY

-   

SECTION: MARTINIZE &amp; INSANE PROTOCOL
6. MARTINI STRUCTURE CONVERSION
From all atom to coarse grained. 

In this tutorial, and as a recommendation, we should use always martinize2 or vermouth method to martinize proteins. Just because it`s easier to specify some characteristics useful for molecular dynamics like "elastic networks" and "position restraints". 

Martinize Proteins
 

 


Output Files. Files that we are going to use later. 
-   
-   



SECTION: MARTINIZE &amp; INSANE PROTOCOL
8. INSANE SYSTEM CONSTRUCTION
Coarse grained system generation. Preparing files for molecular dynamics. 

There are different methods to create a complete system for running molecular dynamics. In general, any of these methods is useful, but the simplest one to avoid having to modify files or use too many commands is to use insane.py to generate the system, adding water molecules and ions. This can be used either to add a membrane to a protein (initially it is intended for this), but in this case we will only add a biological system.
 

Insane System 
 

 

Output Files. Files that we are going to use later. 
-   
-   

SECTION: GROMACS FILE PREPARATIONS
14. DISPOSITION OF FILES IN LYSOZYME_MD FOLDER

 

 

 

- MDP : MDP folder will have every MDP file we will use during the simulation.
- ITP : ITP folder will have every itp file include in topol.top file. 
- Some Files : Single files in the LYZOSYME_MD folder.
- 1.sh : sh script for Dynamics simulation optimization. 

SECTION: GROMACS FILE PREPARATIONS
10. FILES USED IN THIS SECTION

GENERATED IN A PREVIOUS SECTION  :
    --> Insane Output
    --> Insane Output
    --> Martini Output

USED IN THIS SECTION :
   
   
   
   
   
   
   

GENERATED IN THIS SECTION :
   
   

SECTION: GROMACS MOLECULAR DYNAMICS
17. MOLECULAR DYNAMICS GROMACS v2019 - GENERAL ANALYSIS

The analysis of molecular dynamics (MD) simulations in GROMACS is crucial for understanding atomic-level interactions in biomolecular structures. GROMACS provides detailed trajectories, and the analysis reveals valuable insights into dynamics and stability. Aspects such as conformational variability and binding site identification are explored, contributing to the understanding of biological processes. Advanced analytical techniques allow for precise examination of energetics. In summary, GROMACS facilitates a detailed analysis that drives advancements in biomolecular research.

This are the most common analysis for Gromacs simulations. There are others that it can be found in Gromacs-Command. These are attached a simple script for Gromacs analysis just with 3 command RMSD, RMSF and Gyrate. As was said, there are others analysis you would need to use but theses ones function like example of using. 

 


SECTION: PEGylation Method - Polyply v1.0 Methodology
18. FILES USED IN THIS METHODOLOGY

GENERATED IN PREVIOUS SECTIONS :
   --> Clean, just the protein and conects.
   --> Clean, no <Position Restraints> and <Rubber Bands> options.

GENERATED IN THIS SECTION :
   
   
   
   
   
   


SECTION: PEGylation Method - Polyply v1.0 Methodology
19. FILES CREATION PRIOR POLYPLY USAGE - itp and combined_links.ff

Polyply v1.0 is a specialized software designed for the efficient development of polymers within a coarse-grained force field framework. This innovative tool streamlines the process of generating polymer structures, including polymer melts, amorphous blends, and liquid-liquid phase-separated systems. Its primary function revolves around simplifying the creation of input files (itp files) and initial configurations for diverse polymer scenarios. Polyply proves particularly valuable for researchers and scientists engaged in molecular dynamics simulations, offering a user-friendly interface and facilitating the exploration of polymer behaviors at a larger scale.

The polymers as PEG are composed by 3 type of bead.
- Linker bead --> MEE.itp
- Polymer bead --> PEO.itp
- OH bead --> OH_end.itp

If you need more information of what kind of bead are related or why are need it, please visit : PEGylation Proteins Book. They published a book with a methodology in Martini 3 version, methodology that don't work with Polyply v1.0 for martini 2.

The PEO.itp file can be downloaded from Martini Coarse Grained Website, and its has this format
 

The MEE.itp and OH_end.itp files can be found on Martini PEGylation Proteins Book , and its has this format
 

 

<Attached Files >

<Attached Files >

The combined_links.ff file need to be written the first time. This is because there is no single combined_links.ff file, but depends on the structure of the protein you want to PEGylate and the amount of PEG molecules you plan to add to the system. 
For this tutorial we select aminoacid number 1, the first Lysine, other relevant information is Lysozyme has only 287 beads in CG format. 
 
In order not to confuse the reader and since the combined_links.ff file is one of the most relevant files for protein gluing, a notes section is added where each section of the file is explained. In order not to confuse the reader.

 


SECTION: PEGylation Method - Polyply v1.0 Methodology
20. POLYPLY USAGE METHODOLOGY

To generate any protein bound to x number of polymers, 3 commands must be used. All these commands are included in the POLYPLY software. These commands are : polyply gen_seq, polyply gen_params & polyply gen_coords. The first one, polyply gen_seq is in charge of creating the sequences.json file, which corresponds to a file that only takes graphic input in the form of a json file. The second, polyply gen_params, generates an itp file with the parameters of the amino acid sequence plus the PEG molecules we want to add in the place we set earlier. Finally, the last command, polyply gen_coords, will use the coordinates of our coarse grained protein and the previously generated itp file to create a .gro file with the coordinates of our new PEGylated protein.

POLYPLY GEN_SEQ

Recommendation : Before use polyply gen_seq, copy from molecule_0 al lines related with position restraints and rubber bands to other blank text document. Then erase from molecule_0 those lines. Polyply gen_seq always give error when you use it when position restraints and rubber bands. 
 

 

Output Files. Files that we are going to use later. 
   

POLYPLY GEN_PARAMS
 
Output Files. Files that we are going to use later. 
   

POLYPLY GEN_COORDS 

Recommendation
After polyply command, remenber change charge of the bead from 1.0 to 0.0. 
 
Output Files. Files that we are going to use later. 
   

SECTION: MARTINIZE &amp; INSANE PROTOCOL
7. VISUALIZE OUR : MARTINI CONVERSION RESULTS!

The common result of converting an atomistic model to a coarse grained model is a structure made up of spheres or beads in a ratio of 4 to 1 atom per bead.

 

 


SECTION: MARTINIZE &amp; INSANE PROTOCOL
9. VISUALIZE OUR : INSANE CONSTRUCTION RESULTS!

Following the same idea as before, the result of insane is the construction of a box composed of our protein in question, chosen solvent corresponding to water molecules and antifreeze water and finally ions both sodium and chlorine molecules.

 

Obviously, the system will still consist of beads that to the human eye are NOT connected, however, given the parameter files such as itp files, we know that computationally, they ARE. So DO NOT BE AFRAID OF THE PROCESS, later on they will be connected and the expected structure will be displayed.

SECTION: GROMACS MOLECULAR DYNAMICS
16. VISUALIZE OUR : GROMACS MOLECULAR DYNAMICS RESULTS!

Two main results will be obtained. The files production.gro and production.xtc will correspond to the direct results of the molecular dynamics and the files run-connect.pdb and final.xtc, which will correspond to the correction of the direct results of the molecular dynamics, visible to the human eye as structures. 

Both production files will have the normal format of any coarse grained file, so they are not viewable or understandable by the human eye. So the corrected files will be the ones we will use from now on.

 

 


SECTION: PEGylated Protein GROMACS MOLECULAR DYNAMICS
22. As explained above, once we have our pegylated protein structure, we will proceed to the construction of the system. 
For this we used the same steps previously performed, listed below:

Step 8 --> To build the Pegylate system in a water box with ions. Make sure that you add some dimensions for box, changing the old command to this one, this will help to prevent some particles move into the void.

 
Step 11 to 14 --> To organize the folder where we will run our dynamics. We are going to use the same MDP and ITP files used before, with 1 exception, our new lysoPEG.itp file. 
Step 15 --> To run molecular dynamics. We are going to use the same command as before. 
Step 17 --> Analyze molecular dynamics. 
Step 18 --> We erase Position Restraints and Rubber Bands of our molecule_0.itp file. Now those lines we must add again to our lysoPEG.itp file. 

NEW FILES USED IN THIS METHODOLOGY
  
  


SECTION: PEGylation Method - Polyply v1.0 Methodology
21. VISUALIZE OUR : PEGylation Method - Polyply v1.0 Methodology  RESULTS!

Following the same idea as before, the result of PEGylation is a model composed by our protein in question, and MEE linker, PEG beads and OH end.

 


SECTION: PEGylated Protein GROMACS MOLECULAR DYNAMICS
23. VISUALIZE OUR : PEGylated Protein MD RESULTS!

As in step 16. Two main results will be obtained. The files production.gro and production.xtc will correspond to the direct results of the molecular dynamics and the files run-conect.pdb and final.xtc, which will correspond to the correction of the direct results of the molecular dynamics, visible to the human eye as structures. 

Both production files will have the normal format of any coarse grained file, so they are not viewable or understandable by the human eye. So the corrected files will be the ones we will use from now on.

 


SECTION: FINAL REMARKS
24. Finally, the system for creating a coarse grained structure and adding any number of polymers of choice, such as in this case PEGylates, is an efficient way to understand the behavior of any possibly modified structure. 

Anyway, please refer to the official pages of each program for more information.