In our project, since we were targeting the interaction between DENV NS5 and hSTAT2, we needed a structure of their PPI complex. But from our literature survey, we found that the complex structure for DENV NS5-hSTAT2 is not yet experimentally solved yet and in fact, the crystal structure of a part of hSTAT2 was experimentally solved very recently (in 2020). So, we took the structures of DENV NS5 and hSTAT2 from Protein Data Bank and formed the complex docked structure (for more details on this, please visit the Modelling section). This structure (Model 1) can be used (by iGEM and non-iGEM community members) for further work on DENV NS5 and hSTAT2 interaction until the structure is solved experimentally.
Downloadable file: Model
Since the structures available in PDB were only for partial DENV NS5 and hSTAT2. and we needed complex structure of full-length proteins, we first obtained complete structure of DENV NS5 and hSTAT2 by homology modeling and then docked it to get the complex structure (for more details on this, please visit the Modelling section). Hence, this structure (Model 2) can also be used for further work on DENV NS5-hSTAT2 interaction.
Downloadable file: Model
In our project, we generated a library of potential peptide inhibitors which can be used to inhibit the interaction between DENV NS5-hSTAT2 and hence can be further modified for therapeutic or experimental purposes. Due to the time constraint and the multi phase project module, we were unable to characterize them in-vitro using FRaPPe, but we have performed several in-silico characterizations to study their potential as iPEPs. For more details on these aspects please visit the Modelling Section.
Downloadable file: IPep_library
Molecular Dynamic simulation played a vital role in our project. It is not difficult to imagine that running any kind of simulation is replete with challenges and possibilities of errors. We faced these during our work and thus decided to contribute our own pointers to deal with very commonly encountered errors while conducting protein complex simulations. We also recommend checking the GROMACS forum to find answers to your questions.
- Usually when you take large proteins for doing MDS, it is recommended to carry out energy minimization before adding water and ions. This will help you avoid errors such as presence of high energy bonds in the protein complex during energy minimization, post addition of water and ions. In our case we had to carry out energy minimization 3 times before adding water and ions.
- Creating an index file is also recommended before carrying out the steps because creating the desired group in the index file is very much necessary to do analysis.
- Carrying out energy minimization, equilibration and final MD run (depending upon the time steps you have chosen) is quite time-consuming. Hence, it is recommended to use “nohup”(which makes the code run in background) before writing your GROMACS command and saving the steps to a log file.
- After you are done with your final MD run, it’s necessary to remove water molecules from the simulated md.xtc file before loading it into VMD for visualization for which you can use trjconv.
- Before doing visualizing the interaction it’s also recommended that you centre one of your proteins.
- Analysis which can be done to see whether structures of the protein are changing or not during interaction is to check the RMSD plot of the individual proteins.
- To check how the distance changes between the centre of mass of both the proteins, you can use a mindist tool available in gromacs.
- Number of Hydrogen bonds plotted with respect to time can also give a give idea of how stable your protein is.