Team:EPF-Lausanne/Modeling

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= Modeling =
 
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==To do==
 
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;- Model allosteric interactions between LOVTAP & TrpR:
 
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:What will be done:
 
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:- Model of LOVTAP in dark phase
 
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:- Model of LOVTAP in light phase
 
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:- Characterize how the J-alpha helix changes
 
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:- Model sturctural changes that enhance the switch feature of LOVTAP e.g. in dark phase: really weak interaction between LOVTAP and the corresponding DNA sequence, in light phase: strong binding of LOVTAP on DNA.
 
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<html><center>
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<a href="https://2009.igem.org/Team:EPF-Lausanne/Theory" onMouseOver="document.MyImage4.src='https://static.igem.org/mediawiki/2009/thumb/8/83/Theory_nb.jpg/150px-Theory_nb.jpg';" onMouseOut="document.MyImage4.src='https://static.igem.org/mediawiki/2009/thumb/c/c9/Theory.jpg/150px-Theory.jpg';">
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<img src="https://static.igem.org/mediawiki/2009/thumb/c/c9/Theory.jpg/150px-Theory.jpg" name="MyImage4"></a>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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==Modeling reference==
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<a href="https://2009.igem.org/Team:EPF-Lausanne/Implementation" onMouseOver="document.MyImage5.src=' https://static.igem.org/mediawiki/2009/thumb/3/3c/Impl_nb.png/150px-Impl_nb.png';" onMouseOut="document.MyImage5.src='https://static.igem.org/mediawiki/2009/thumb/6/65/Impl.png/150px-Impl.png';">
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===LOVTAP simulation===
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<img src="https://static.igem.org/mediawiki/2009/thumb/6/65/Impl.png/150px-Impl.png" name="MyImage5"></a>
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<span style="color:midnightblue">We will follow the following article protocol: </span>
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<br> Freddolino, P.L., Dittrich M., Schulten K., Dynamic Switching Mechanisms in LOV1 and LOV2 Domains of Plant Phototropins. Biophysical Journal, 91, 3630-3639, 2006 ([http://www.ncbi.nlm.nih.gov/sites/entrez?db=pmc&cmd=search&term=PMC1630464 Pubmed])
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===VMD informations===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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VMD is used to visualize molecules. It is quite user friendly.
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* A tutorial for VMD can be found [http://www.ks.uiuc.edu/Training/Tutorials/ here].
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===NAMD informations===
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<a href="https://2009.igem.org/Team:EPF-Lausanne/Analysis" onMouseOver="document.MyImage6.src='https://static.igem.org/mediawiki/2009/thumb/0/0c/Analysis_nb.jpg/150px-Analysis_nb.jpg';" onMouseOut="document.MyImage6.src='https://static.igem.org/mediawiki/2009/thumb/8/84/Analysis.jpg/150px-Analysis.jpg';">
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NAMD performs minimization and equilibration.
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<img src="https://static.igem.org/mediawiki/2009/thumb/8/84/Analysis.jpg/150px-Analysis.jpg" name="MyImage6"></a></center>
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* A tutorial is on the same page as for VMD, [http://www.ks.uiuc.edu/Training/Tutorials/ here].
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</html>
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* [http://www.ks.uiuc.edu/Research/namd/2.7b1/ug/ NAMD 2.7b1 User's Guide]
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<br>
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<br>
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===Run a simulation===
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<html><center>
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A simulation is composed of different steps. Here are a few links that deal with heating and stabilization.
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<font size="6" color="#007CBC"><i>Modeling overview</i></font>
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* [http://www.ks.uiuc.edu/Research/namd/tutorial/NCSA2002/hands-on/ Building Gramicidin A: Equilibration]: protocol uses a single .conf file, heating process is too fast.
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</center></html>
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* [http://www.biochemistry.uab.edu/robinson/documents/howto/modeling/NAMD_notes.htm NAMD notes from Robinson Lab]: a really nice heating process, but involves different .conf files, which is really painful.
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<br>
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----
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<br>
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==Protein domain of interest==
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Our protein of interest is [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP LOVTAP]. This protein was sythetically engineered by [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=18667691 Sosnick] group. It is a fusion protein between a LOV domain (Avena Sativa phototropin 1) and the E. Coli tryptophan repressor.
 +
This protein undergoes changes under light activation as shown by Sosnick et al, in fact when the protein is activated by light it binds DNA and inversely.
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For more information about LOVTAP protein please [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP click here].
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<br><br>
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==Goal==
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<br><br>
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==Starting material==
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Both LOV domain crystallography files were obtained from [http://www.rcsb.org/pdb/home/home.do RCSB]:
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:[http://www.rcsb.org/pdb/explore/explore.do?structureId=2V0W Light activated LOV domain]
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:[http://www.rcsb.org/pdb/explore/explore.do?structureId=2V0U Dark LOV domain]
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==Implementation of the simulation==
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These crystallographies were done by [http://www.ncbi.nlm.nih.gov/pubmed/18001137 Halavaty et al.].
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LOV domains are the light-sensitive portion of phototropins. They absorb light through a flavin cofactor, photo-chemicaly form a covalent bond between the chromophore and a cysteine residue in the protein, and proceed to mediate activation of an attached kinase domain.
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<br><br>
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==Molecular dynamics: a little theory==
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===Generating input files===
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Molecular dynamics simulation consists of the numerical, step-by-step, solution of the classical equations of motion. For this purpose we need to be able to calculate the forces acting on the atoms, and these are usually derived from a potential energy.  
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First we need a compatible .pdb in addition to parameter and topology files. Steps to generate all the input files are explained in detail on this page [[Team:EPF-Lausanne/Modeling/Simulation|How to generate input files]]. This is a kind of summary of the tuto.
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===.conf parameters===
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<html>
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We should explain here what are the keywords we use in the .conf.
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<script type="text/javascript" language="JavaScript"><!--
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function HideContent(d) {
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document.getElementById(d).style.display = "none";
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}
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function ShowContent(d) {
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document.getElementById(d).style.display = "block";
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}
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function ReverseDisplay(d) {
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if(document.getElementById(d).style.display == "none") { document.getElementById(d).style.display = "block"; }
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else { document.getElementById(d).style.display = "none"; }
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}
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//--></script>
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===Run a complete simulation===
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<p>
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We start from .pdb, .psf, .rtf generated in the previous section. Complete process is on a separate page [[Team:EPF-Lausanne/Modeling/RunSimulation|How to run a simulation]].
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<a href="javascript:ReverseDisplay('hs1')">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Click here to expand</a>
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</p>
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<div id="hs1" style="display:none;">
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<p>
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This potential energy can be divided into:
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<h3>The non-bonded interactions:</h3>
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<li>The <i>Lennard-Jones potential</i> is the most commonly used form, with two parameters: σ, the diameter, and ε, the well depth. It takes into account the Van der Waals forces. It represents the non-bonded forces and the total potential energy can be calculated from the sum of energy contributions between pairs of atoms.
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<center>
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<img src="https://static.igem.org/mediawiki/2009/d/da/Lennard_jones_vdw_forces.jpg">
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<img src="https://static.igem.org/mediawiki/2009/thumb/f/f1/Lennard_jones.jpg/300px-Lennard_jones.jpg">
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==Molecular dynamics theory==
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<center><i><u>Lennard-Jones pair potential showing the r<sup>−12</sup> and r<sup>−6</sup> contributions</u></i></center>
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</center></li>
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<br>
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<li>when electrostatic charges are present, we add the <i>Coulomb force</i>, where Q1, Q2 are the  charges and ϵ0 is the permittivity of free space
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<center><img src="https://static.igem.org/mediawiki/2009/thumb/4/42/Coulomb.jpg/200px-Coulomb.jpg"></center>
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</li>
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<br><br><br>
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<h3>The bonded interactions:</h3>
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Angles, bonds and dihedral angles have to be taken into account:
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<br>
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<center><img src="https://static.igem.org/mediawiki/2009/thumb/2/28/Bonded.jpg/400px-Bonded.jpg"></center>
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<br><br>
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Molecular dynamics simulation consists of the numerical, step-by-step, solution of the classical equations of motion. For this purpose we need to be able to calculate the forces acting on the atoms, and these are usually derived from a potential energy. This potential energy can be divided into:
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To understand a bit more, you can see the following article:
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* '''the non-bonded interactions''':
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<a href="https://static.igem.org/mediawiki/2009/3/3e/Introduction_to_molecular_Dynamics_Simulation.pdf">Introduction to Molecular Dynamics Simulation - Michael P. Allen</a>
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**The ''Lennard-Jones potential'' is the most commonly used form, with two parameters: σ, the diameter, and ε, the well depth. It takes into account the Van der Waals forces. It represents the non-bonded forces and the total potential energy can be calculated from the sum of energy contributions between pairs of atoms.
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[[Image:lennard_jones_vdw_forces.jpg|frame|center|Lennard Jones potential ]]
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** when electric charges are present, we add the ''Coulomb force''
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* '''the bonded interactions''':
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<p align="center" class="style1"><a href="#top"><img src="https://static.igem.org/mediawiki/2009/thumb/0/06/Up_arrow.png/50px-Up_arrow.png" alt="Back to top" border="0"></a><br></p>
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<br>
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</p>
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</div>
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</html>
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<br><br>
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==Steps==
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===Minimization===
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===Equilibration===
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===Analysis and validation===
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===Simulation===
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===Atom movement analysis===
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<br><br>
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==References==
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<br><br>
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==Analysis methodology==
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<br><br>
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==Results==
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<br><br>
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The protein moves thanks to different forces, which can be separated in bonded forces and non-bonded forces. The bonded forces correspond to
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==To do==
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* the ''Lennard-Jones potential'',
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;- Model allosteric interactions between LOVTAP & TrpR:
 +
:What will be done:
 +
:- Model of LOVTAP in dark phase
 +
:- Model of LOVTAP in light phase
 +
:- Characterize how the J-alpha helix changes
 +
:- Model structural changes that enhance the switch feature of LOVTAP e.g. in dark phase: really weak interaction between LOVTAP and the corresponding DNA sequence, in light phase: strong binding of LOVTAP on DNA.
 +
;- Between Light state and Dark State
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:- RMS between light state and dark state
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:- length between the two arms N C
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:- only cytochrome with interactions in light state and dark state
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<br><br>
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[[Image:Coulomb.jpg‎|frame|center|Coulomb force ]]
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==To envisage ==
==To envisage ==
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<br>- Molecular mutationnal assay
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<br>- Molecular mutational assay
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==Already done==
 
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Here is our first movie from the modeling, showing the behavior of the protein in the dark state condition:
 
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[[Media:2v0u_dark_state.mov | Dark State]]
 
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After having modified some parameters in the parameter files, here is our second movie, concerning the light state of the protein this time, with the FMN:
 
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[[Media:Light_FMN_without_water.mov‎  | Light State with FMN without water]]
 
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Latest revision as of 08:34, 21 September 2009



Contents

                               


Modeling overview


Protein domain of interest

Our protein of interest is LOVTAP. This protein was sythetically engineered by Sosnick group. It is a fusion protein between a LOV domain (Avena Sativa phototropin 1) and the E. Coli tryptophan repressor. This protein undergoes changes under light activation as shown by Sosnick et al, in fact when the protein is activated by light it binds DNA and inversely. For more information about LOVTAP protein please click here.

Goal



Starting material

Both LOV domain crystallography files were obtained from RCSB:

Light activated LOV domain
Dark LOV domain

These crystallographies were done by Halavaty et al..

Molecular dynamics: a little theory

Molecular dynamics simulation consists of the numerical, step-by-step, solution of the classical equations of motion. For this purpose we need to be able to calculate the forces acting on the atoms, and these are usually derived from a potential energy.

         Click here to expand



Steps

Minimization

Equilibration

Analysis and validation

Simulation

Atom movement analysis



References



Analysis methodology



Results



To do

- Model allosteric interactions between LOVTAP & TrpR
What will be done:
- Model of LOVTAP in dark phase
- Model of LOVTAP in light phase
- Characterize how the J-alpha helix changes
- Model structural changes that enhance the switch feature of LOVTAP e.g. in dark phase: really weak interaction between LOVTAP and the corresponding DNA sequence, in light phase: strong binding of LOVTAP on DNA.
- Between Light state and Dark State
- RMS between light state and dark state
- length between the two arms N C
- only cytochrome with interactions in light state and dark state



To envisage


- Molecular mutational assay



Retrieved from "http://2009.igem.org/Team:EPF-Lausanne/Modeling"