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Tutorial 8

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NedaRahmani edited this page Feb 14, 2025 · 22 revisions

Tutorial 8: Add More Proteins and Place Proteins based on Curvature

The files needed for the tutorial can be manually downloaded here.

This tutorial is divided into two parts. In the first part you will use the input files from tutorial 4 to build a system and easily add more proteins to the system. Whereas, in the second part you build a curved membrane and place the proteins based on curvature (similarly to tutorial 7).

8-1 Add more proteins

Step 1: Generate point folder Use the following command to generate the point folder.

TS2CG PLM -TSfile Sphere.tsi -bilayerThickness 3.8 -rescalefactor 4 4 4  -o point_vesicle

Since you are using the input files from Tutorial 4, the output from PLM will contain three proteins in total, two of type 1 and one protein of type 2. This can be seen in point/IncData.dat.

< Inclusion NoInc       3   >
< id typeid pointid lx ly lz  >
           0      1      5      0.499      -0.864      0.059
           1      1      22     0.481      -0.765     -0.429
           2      2      30    -0.177      -0.945      0.275

Step 2: Add more proteins

In tutorial 4 you would now use TS2CG PCG to build the system but in this tutorial, you include one more step, TS2CG INU. Use TS2CG INU -h for available flags. This function will modify the point folder by adding more proteins of your choice. For example, to add three proteins of type 1 in the outer leaflet you can run the following command:  

TS2CG INU -p point_vesicle -n 3 -t 1 -r 5 -o point_vesicle_new -l outer

This command will modify point folder and save the updated point folder as point_new.

Step 3: Add more proteins of a different type

Now we also want to add some of protein of type 2 by modifying the point_new. Execute the following command:

TS2CG INU -p point_vesicle_new -n 2 -t 2 -r 5 -o point_vesicle_new2 -l outer

NOTE: Different values of the –-radius flag will alter the number of available positions to place proteins, larger radius will result in fewer protein positions.

This command will modify point_new by adding two more proteins of type 2 in the outer leaflet and create a updated folder called point_new2 containing the total amount of proteins. In total the system now contains five proteins of type 1 and three proteins of type 2. Number of proteins and corresponding type can be seen in point_new2/IncData.dat.

< Inclusion NoInc 8 >
< id typeid pointid lx ly lz >
           0            1            5    0.499   -0.864    0.059
           1            1           22    0.481   -0.765   -0.429
           2            2           30   -0.177   -0.945    0.275
           3            1         7030    1.000    0.000    0.000
           4            1          709    1.000    0.000    0.000
           5            1          241    1.000    0.000    0.000
           6            2         5382    1.000    0.000    0.000
           7            2         1555    1.000    0.000    0.000

Step 4: Build a bilayer vesicle with proteins based on modified point folder using PCG

Finally, in this step you will use PCG as done in previous tutorials but with point_new2 provided by INU in the previous step.

TS2CG PCG -str input_vesicle.str -Bondlength 0.2 -LLIB "./files/Martini3.LIB" -dts ./point_vesicle_new2 -incdirtype Local -defout system_vesicle

The system can now be visualized using VMD or similar software, where the eight proteins can be seen as part of the membrane.

A script named run_tut8_Vesicle.sh is available in the tut8/outputs/part1 folder. This script will execute the commands presented above and it will run the TS2CG output using GROMACS.

  1. Energy Minimization without Solvent: Conduct a standard energy minimization, excluding solvent from the system.

  2. Short Equilibration without Solvent: Run a brief equilibration step without solvent.

tut8-1 Fig. 8-1. Initial structure of the created POPC vesicle with eight proteins (left), and the vesicle after a brief vacuum simulation using GROMACS (right). Visualized with VMD.

8-2: Place proteins based on curvature

Step 1: Generate point folder

As in part I you start by generating a point folder but this time of a curved membrane. Execute the following command:

TS2CG PLM -TSfile prep/Small_Curved.tsi -bilayerThickness 3.8 -rescalefactor 4 4 4

Step 2: Add proteins based on curvature

In this tutorial you will place the proteins in the negatively curved region by using the following command. You are placing 10 proteins of type 1 where the curvature is negative and we amplify the placement by setting k to a large number namely, 100, and the proteins will be placed in both leaflets.

Execute INU by using the following command:

TS2CG INU -p point_SmallCurved -n 10 -t 1 -r 5 -c -0.5 -k 100 -o point_SmallCurved_new -l both

The modifications will be saved in a new point folder called point_new.

Step 3: Build a curved bilayer membrane based on the modified point folder using PCG

Step 4: Build a bilayer based on modified point folder using PCG

Finally, build the bilayer by executing the following command:

TS2CG PCG -str input_SmallCurved.str -Bondlength 0.2 -LLIB ./files/Martini3.LIB -dts ./point_SmallCurved_new -incdirtype Local -defout system_SmallCurved

You can now visualize the system using VMD or similar software, where you see a curved membrane with proteins in or close to the negatively curved region.

tut8-2 Fig. 8-2. Initial structure of the bilayer membrane with proteins positioned in negatively curved regions (left), and the system after a brief vacuum simulation using GROMACS (right). Visualized with VMD.

A script named run_tut8_SmallCurved.sh is available in the tut8/outputs/part2 folder. This script will execute the commands presented above and it will run the TS2CG output using GROMACS.

1. Energy Minimization without Solvent: Conduct a standard energy minimization, excluding solvent from the system.

2. Short Equilibration without Solvent: Run a brief equilibration step without solvent.  

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