I have been successful in generating the homology model of the catalytic region of a chitin deacetylase protein but am facing some difficulties while simulating the protein. (My protein has Cobalt as a metal in the active site residue)I would like to perform the simulation to minimize the energy for the protein and bring some disallowed residues within the favorable region before I dock the substrate (chitin). Although I am getting an equilibrated structure after simulation (Analysed through RMSD etc. plots) but I am unable to bring any of the residues within the favorable ones. I have performed 5ns simulation for the protein, but the problem still exists. Another observation was that the protein structure (PDB file generated from the last frame after simulation) deviated to a great extent from the original structure after the simulation, hence the catalytic region is also getting deviated. So the residues that should bind with the substrate is unable to bind (When compared with the template structure). My ultimate aim is to perform a docking study of the enzyme with its substrate.
The attached paper shows a similar workflow where a 3 ns simulation was able to bring all the disallowed residues into favourable regions.
I would request you to please help me with your valuable suggestions that would guide me in the right way.
You can first perform a side-chain modeling or loop building of the modeled protein and then subject to energy minimization.
Please check if the forcefield you used has parameters for Cobalt.
If there are previous reports that the template and your modelled protein have similar active site region, you can go for only energy minimization.
Also I am more concerned about the metal atom, it will be interesting to see how it is behaving during the simulations, as you have said that the structure has deviated.
Best wishes !!
Well i have some more suggestions.. After obtaining your generated model and its validation, you need to know where exactly you want to dock your ligand. If you don't know the active site of your protein submit it to Dogsite scorer.. It will give you some pockets. Select the one with score ranging from 0.6 to 0.8. The one with score more than 0.8 is preferably the best pocket to be used.
Sometimes the active site of the Template is also provide you useful information.
Residues in disallowed region mostly due inappropriate template used. so select the template on the basis of E-value, Sequence query and identity. If u don't find any suitable template use I-Tasser server building a model.
http://zhanglab.ccmb.med.umich.edu/I-TASSER/
@Sohaib: We used I-Tasser for generating model.
@Jamal: We do have an idea on the docking site. We have done so. Now the docking energy to the structure obtained after 5ns simulation of the apo metalloprotein is coming acceptable (~-5kcal/mol). However the disallowed residues remain disallowed.
Any suggestions?
I think, you should go for selective minimisation of outliers by putting a conformation constraints. In this way, somehow you would able to minimise the outliers only, rather whole protein and conformational of protein unaffected then and also would assist outliers to adjust their Phi and Psi angles (Note: sometimes it is possible that you still get outliers thereafter the minimisation). After that, dock the co-factor (cobalt) in its pocket or if it has some other co-enzyme binding then try to dock that co-enzyme (may be any vitamin etc). Later on if in case it is co-enzyme try to evaluate your docking simulated model for conserved residue interactions with the co-enzyme and finally if you have conserved interactions then you could go for any other ligand based docking.
may be this paper helps you.
https://www.researchgate.net/publication/248382055_Cystathionine__Lyase_like_Protein_with_Pyridoxal_Binding_Domain_Characterized_in_Leishmania_Major_by_Comparative_Sequence_Analysis_and_Homology_Modelling
Article Cystathionine β Lyase like Protein with Pyridoxal Binding Do...
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