Difference between revisions of "Team:Queens Canada/Modeling"
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<li><a href="https://2015.igem.org/Team:Queens_Canada/Attributions">Attributions</a></li> | <li><a href="https://2015.igem.org/Team:Queens_Canada/Attributions">Attributions</a></li> | ||
<li><a href="https://2015.igem.org/Team:Queens_Canada/Safety">Safety</a></li> | <li><a href="https://2015.igem.org/Team:Queens_Canada/Safety">Safety</a></li> | ||
− | <li><a href="https://2015.igem.org/Team:Queens_Canada/Practices">Human Practices</a></li> | + | <li><a href="https://2015.igem.org/Team:Queens_Canada/Practices">Human Practices</a> |
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+ | <li><a href="https://2015.igem.org/Team:Queens_Canada/Practices/Academics">Academics</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:Queens_Canada/Practices/Outreach">Community Outreach</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:Queens_Canada/Practices/IP">Intellectual Property</a></li> | ||
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<li><a href="https://2015.igem.org/Team:Queens_Canada/Notebook">Notebook</a></li> | <li><a href="https://2015.igem.org/Team:Queens_Canada/Notebook">Notebook</a></li> | ||
<li><a href="https://2015.igem.org/Team:Queens_Canada/Parts">Parts</a></li> | <li><a href="https://2015.igem.org/Team:Queens_Canada/Parts">Parts</a></li> | ||
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<li><a href="https://2015.igem.org/Team:Queens_Canada/Background">Background</a></li> | <li><a href="https://2015.igem.org/Team:Queens_Canada/Background">Background</a></li> | ||
<li><a href="https://2015.igem.org/Team:Queens_Canada/Modeling">Modeling</a></li> | <li><a href="https://2015.igem.org/Team:Queens_Canada/Modeling">Modeling</a></li> | ||
− | <li><a href="https://2015.igem.org/Team:Queens_Canada/AFP_Scaffold"> | + | <li><a href="https://2015.igem.org/Team:Queens_Canada/AFP_Scaffold"> The Ice Queen</a></li> |
− | <li><a href="https://2015.igem.org/Team:Queens_Canada/Circ_AFP"> | + | <li><a href="https://2015.igem.org/Team:Queens_Canada/Circ_AFP"> Icefinity</a></li> |
</ul> | </ul> | ||
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<div class="intro"> | <div class="intro"> | ||
− | <h1> | + | <h1>MODELING: INTRODUCTION</h1> |
− | <p>How many times have you gone to do something, put your heart and soul into it and then found out it hasn't worked? Your hours of laborious effort turned all for | + | <p>How many times have you gone to do something, put your heart and soul into it and then found out it hasn't worked? Your hours of laborious effort turned all for nothing? Us too and this year, we set out to avoid this very dilemma, or at least to try and minimize its effects on our project.</p> |
− | <p>The modeling process was used to gain an understanding of what we expected from the wet lab | + | <p>The modeling process was used to gain an understanding of what we expected from the wet lab work. The principle behind design was to troubleshoot and optimize the engineered components through simulations to identify mistakes within the theoretical space before using time and resources in the lab. </p> |
<p>This preliminary design can be divided into two components: modeling a circular AFP and scaffold design. </p> | <p>This preliminary design can be divided into two components: modeling a circular AFP and scaffold design. </p> | ||
</div> | </div> | ||
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<div id="modelbodytext"> | <div id="modelbodytext"> | ||
− | <h1> | + | <h1>MODELING A CIRCULAR AFP</h1> |
+ | <h2 align="center"><a href="https://2015.igem.org/Team:Queens_Canada/Circ_AFP">Icefinity</a></h2> | ||
<figure style="float: left; width: 400px;"> | <figure style="float: left; width: 400px;"> | ||
<img src="https://static.igem.org/mediawiki/2015/1/14/Qqq_QGEM_Type3AFP.jpg" style="width: 400px;"/> | <img src="https://static.igem.org/mediawiki/2015/1/14/Qqq_QGEM_Type3AFP.jpg" style="width: 400px;"/> | ||
− | <figcaption>Figure 1. <strong>Crystal structure of the Type III Ocean Pout.</strong>This protein is represented by the PDB file 1AME. The distance between the two termini was found using Pymol<sup>1</sup>.</figcaption> | + | <figcaption>Figure 1. <strong>Crystal structure of the Type III Ocean Pout.</strong>This protein is represented by the PDB file 1AME. The distance between the two termini was found using <a href="https://www.pymol.org/">Pymol</a><sup>1</sup>.</figcaption> |
</figure> | </figure> | ||
− | <p style="margin-top: 120px;">After | + | <p style="margin-top: 120px;">After research different antifreeze proteins, we decided to work with Type III AFP, from the ocean pout. Relatively active in antifreeeze activity, it serves as an ideal AFP for use in industrial purposes. Furthermore, its termini are only 19.8 angstroms apart, making it easier to circularize using a smaller linker (Figure 1).</p> |
<p>The modeling process for the circularization of an antifreeze protein can be explained by a three-step approach to protein design (seen below). </p> | <p>The modeling process for the circularization of an antifreeze protein can be explained by a three-step approach to protein design (seen below). </p> | ||
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<h2>Linker Design & Spatial Fitting</h2> | <h2>Linker Design & Spatial Fitting</h2> | ||
− | <p><strong>Approach 1:</strong> The idea for this project was meant to be a continuation of the work of team <a href="https://2014.igem.org/Team:Heidelberg">Heidelberg 2014</a> and validation of their parts. Using their <a href="http://parts.igem.org/Part:BBa_K1362000">intein BioBrick </a> we wanted to circularize our own protein, a Type III AFP. Using the CRAUT software | + | <p><strong>Approach 1:</strong> The idea for this project was meant to be a continuation of the work of team <a href="https://2014.igem.org/Team:Heidelberg">Heidelberg 2014</a> and validation of their parts. Using their <a href="http://parts.igem.org/Part:BBa_K1362000">intein BioBrick </a> we wanted to circularize our own protein, a Type III AFP. Using the <a href="https://github.com/igemsoftware/Heidelberg_2014">CRAUT software</a>, we ran our protein through the program. After fixing syntax errors, the following linkers were suggested:</p> |
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− | <p> | + | <p>The sequences in <span id="red">red</span> represent the extein sequences (scars that will be left after the intein reaction), and the sequences in <span id="purple">purple</span> represent a secondary structure (alpha-helix). Preliminary spatial evaluation using PyMOL suggested these were not ideal for linking the termini of our protein of interest. For the purpose of progression, we adapted the second sequence and opted for running simulations on our own linker designs. </p> |
<p><strong>Approach 2:</strong>Using the extein sequence tested by Heidelberg 2014, linkers were built within PyMOL and Coot to effectively joined the N- and C- termini. Flexible linkers were chosen to enable the termini to arrange such that the protein remains in its functional conformation. </p> | <p><strong>Approach 2:</strong>Using the extein sequence tested by Heidelberg 2014, linkers were built within PyMOL and Coot to effectively joined the N- and C- termini. Flexible linkers were chosen to enable the termini to arrange such that the protein remains in its functional conformation. </p> | ||
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<br> | <br> | ||
− | <h1>AFP- | + | <h1>AFP-SCAFFOLD DESIGN</h1> |
+ | <h2 align="center"><a href="https://2015.igem.org/Team:Queens_Canada/AFP_Scaffold">The Ice Queen</a></h2> | ||
<figure style="float: right; width: 500px;"> | <figure style="float: right; width: 500px;"> | ||
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<h2>The E/K-coil system</h2> | <h2>The E/K-coil system</h2> | ||
− | <p>We sought a strong, highly efficient means to connecting AFPs to the self-assembling scaffold. After considering our options, we opted for the E/K coil method used by <a href="https://2013.igem.org/Team:Calgary">Calgary iGEM 2013 </a>. These non-covalent interactions are highly specific and should enable selective binding between AFPs and the scaffold subunit. Read more about coiled-coils on our <a href=" | + | <p>We sought a strong, highly efficient means to connecting AFPs to the self-assembling scaffold. After considering our options, we opted for the E/K coil method used by <a href="https://2013.igem.org/Team:Calgary">Calgary iGEM 2013</a>. These non-covalent interactions are highly specific and should enable selective binding between AFPs and the scaffold subunit. Read more about coiled-coils on our <a href="https://2015.igem.org/Team:Queens_Canada/Background">Background page.</a> </p> |
<p> The coil sequences to be used:</p> | <p> The coil sequences to be used:</p> | ||
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− | <p>Upon examination of their work and the sequences used, we noted a discrepancy in their data. According to the PDB file and NMR structure elicited in 2004 <sup>5</sup>, the coils interact in a parallel fashion, incorrectly identified as | + | <p>Upon examination of their work and the sequences used, we noted a discrepancy in their data. According to the PDB file and NMR structure elicited in 2004 <sup>5</sup>, the coils interact in a parallel fashion, incorrectly identified as antiparallel in the registry: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189010">K coil</a> and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189011">E coil.</a> This has now been reviewed by us to indicate the correct interaction according to the PDB file 1U0I (Take a look at the experience pages for both the <a href="http://parts.igem.org/Part:BBa_K1189011:Experience">E coil</a> and <a href="http://parts.igem.org/Part:BBa_K1189010:Experience">K coil</a> to read about our changes). </p> |
<figure style="float: left; width: 580px;"> | <figure style="float: left; width: 580px;"> | ||
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<figcaption>Figure 6. <strong>Theoretical AFP-Scaffold Complex.</strong> Parallel coiled coil interactions selectively attach the AFP to the 12-mer scaffold. The blue ends of the coils represent the N-terminus, and the red the C-terminus.</figcaption> | <figcaption>Figure 6. <strong>Theoretical AFP-Scaffold Complex.</strong> Parallel coiled coil interactions selectively attach the AFP to the 12-mer scaffold. The blue ends of the coils represent the N-terminus, and the red the C-terminus.</figcaption> | ||
</figure> | </figure> | ||
− | <p>To generate a model of our complex, the E-coil was fused to the Type | + | <p>To generate a model of our complex, the E-coil was fused to the Type III AFP and the K-coil was fused to the scaffold subunits. The expected interaction is a parallel alignment as depicted in Figure 6. The introduction of a flexible region between the C-terminus of the scaffold subunit and the coil is introduced to allow the coil movement to enable an interaction to occur without steric hindrance from the protein units. </p> |
<h2>Coiled-Coil Stability Simulations</h2> | <h2>Coiled-Coil Stability Simulations</h2> | ||
<p>Before using these coils we sought out to test their stability and method of interaction in its native form. The coils selected for our project is represented by the PDB file 1U0I, an NMR-solved crystal structure. These coils are described to interact in a parallel fashion (Figure 7a). To simulate this interaction, the individual coils were solvated and run thorugh MD simulations in GROMACS. </p> | <p>Before using these coils we sought out to test their stability and method of interaction in its native form. The coils selected for our project is represented by the PDB file 1U0I, an NMR-solved crystal structure. These coils are described to interact in a parallel fashion (Figure 7a). To simulate this interaction, the individual coils were solvated and run thorugh MD simulations in GROMACS. </p> | ||
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<div id="Stabilityanalysis"> | <div id="Stabilityanalysis"> | ||
<h2>Stability Analysis</h2> | <h2>Stability Analysis</h2> | ||
− | <p>Comparison of the parallel and antiparallel coiled coil interaction was performed with consideration to both the | + | <p>Comparison of the parallel and antiparallel coiled coil interaction was performed with consideration to both the Lennard Jones and Coulomb potential energies as calculated using GROMACS software. The Lennard-Jones potential is a mathematical model that approximates the intermolecular forces between two molecules <sup>6</sup> and Coulomb potential describes the interaction between point charges. Statistical analysis was carried out on the energy output for each file and can be found summarized in Table 2 below.</p> |
<table> | <table> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Lennard-Jones</td> |
<td>-131.8912492</td> | <td>-131.8912492</td> | ||
<td>-132.159454</td> | <td>-132.159454</td> | ||
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<tr> | <tr> | ||
− | <td> | + | <td>Lennard-Jones</td> |
<td>-147.8004057</td> | <td>-147.8004057</td> | ||
<td>-149.036652</td> | <td>-149.036652</td> | ||
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</table> | </table> | ||
<br> | <br> | ||
− | <p>These values suggest that when compared with | + | <p>These values suggest that when compared with the Lennard-Jones potential, the antiparallel orientation is more stable while the Coulomb potential suggests the parallel is slightly more stable. This discrepancy creates uncertainty in the theoretical knowledge of preferred orientation for these engineered coils. For the basis of this project, it is presumed that the NMR structure as determined by <a href="http://www.rcsb.org/pdb/explore.do?structureId=1u0i"> Lindhout et al. </a> is the preferred orientation and thus the coils should interact in a parallel orientation. |
</p> | </p> | ||
</div> | </div> | ||
<div id="Dockingstuff"> | <div id="Dockingstuff"> | ||
− | <h1> | + | <h1>PYROSETTA DOCKING & LINKER TESTING</h1> |
<h2>AFP & Scaffold Docking</h2> | <h2>AFP & Scaffold Docking</h2> | ||
− | <p>In order to test the self-assembly of the AFPs and scaffold proteins with E/K coils, docking simulations were run. These were used to assess the energetic stability of the coiled coil interaction and determine the orientation of the ice-binding surface of the AFP. This was done using <a href="">PyRosetta</a> by following the standard procedure for initial low resolution docking prior to high resolution docking on favourable protein structures. Sorting of low energy dockings was used with consideration given to the proximity of the E/K coils to choose a final selection of proteins for refinement and scoring. The general procedure used is outlined in Figure 8 and the final docked structure reached shown in Figure 9.</p> | + | <p>In order to test the self-assembly of the AFPs and scaffold proteins with E/K coils, docking simulations were run. These were used to assess the energetic stability of the coiled coil interaction and determine the orientation of the ice-binding surface of the AFP. This was done using <a href="http://www.pyrosetta.org/">PyRosetta</a> by following the standard procedure for initial low resolution docking prior to high resolution docking on favourable protein structures. Sorting of low energy dockings was used with consideration given to the proximity of the E/K coils to choose a final selection of proteins for refinement and scoring. The general procedure used is outlined in Figure 8 and the final docked structure reached shown in Figure 9.</p> |
<figure> | <figure> | ||
− | <img src=" | + | <img src="https://static.igem.org/mediawiki/2015/0/01/Qqq_QGEM_AFPscaffdocking.jpg" /> |
− | <figcaption>Figure 8. <strong>Flow chart of general protocol used to determine configuration of docked AFP/scaffold complex.</strong> Flow chart starts with PDB files and structures of the proteins of interest, and involves isolating the most stable conformations for further analysis | + | <figcaption>Figure 8. <strong>Flow chart of general protocol used to determine configuration of docked AFP/scaffold complex.</strong> Flow chart starts with PDB files and structures of the proteins of interest, and involves isolating the most stable conformations for further analysis; <a href="http://www.rcsb.org/pdb/explore.do?structureId=4EGG">T3-10 Scaffold</a> and <a href="http://www.rcsb.org/pdb/explore.do?structureId=1AME">Type III AFP</a>. </figcaption> |
</figure> | </figure> | ||
<figure> | <figure> | ||
− | <img src=" | + | <img src="https://static.igem.org/mediawiki/2015/8/86/Qqq_QGEM_AFPscaff.jpg" /> |
<figcaption>Figure 9. <strong>Final alignment of AFP and scaffold coiled-coil interaction after docking and refinement.</strong>Scaffold subunits are shown in yellow with a rainbow K-coil to demonstrate directionality. An AFP is shown in grey and the ice binding residues are highlighted in orange. </figcaption> | <figcaption>Figure 9. <strong>Final alignment of AFP and scaffold coiled-coil interaction after docking and refinement.</strong>Scaffold subunits are shown in yellow with a rainbow K-coil to demonstrate directionality. An AFP is shown in grey and the ice binding residues are highlighted in orange. </figcaption> | ||
</figure> | </figure> | ||
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<h2>Circularization Program</h2> | <h2>Circularization Program</h2> | ||
− | <p>In order to run molecular dynamic simulations of the circularized AFP, the linkers were individually fit with the scar sequence to optimize positioning to allow the terminal nitrogen and carbon groups in bond length proximity (approximately 1.3 angstroms). To save time and energy in spatially testing multiple linkers, a script was written to utilize PyRosetta’s capabilities to circularize the protein for easy visual assessment of atom positioning. This involved using the small mover method in PyRosetta to allow each movement to be checked in ‘fitting’ the linker to ensure the dihedral angles were within acceptable ranges. The script for this program can be located <a href="">here.</a></p> | + | <p>In order to run molecular dynamic simulations of the circularized AFP, the linkers were individually fit with the scar sequence to optimize positioning to allow the terminal nitrogen and carbon groups in bond length proximity (approximately 1.3 angstroms). To save time and energy in spatially testing multiple linkers, a script was written to utilize PyRosetta’s capabilities to circularize the protein for easy visual assessment of atom positioning. This involved using the small mover method in PyRosetta to allow each movement to be checked in ‘fitting’ the linker to ensure the dihedral angles were within acceptable ranges. The script for this program can be located <a href="https://github.com/dragoschiriac/pyRosettaDocking">here.</a></p> |
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− | <h1> | + | <h1>REFERENCES</h1> |
<p>1. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. </p> | <p>1. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. </p> | ||
<p>2. Chao et al. (1994). "Structure-function relationship in the globular type III antifreeze protein: Identification of a cluster of surface residues required for binding to ice". Protein Science. 3(10):1760-1769.</p> | <p>2. Chao et al. (1994). "Structure-function relationship in the globular type III antifreeze protein: Identification of a cluster of surface residues required for binding to ice". Protein Science. 3(10):1760-1769.</p> | ||
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Latest revision as of 16:08, 18 September 2015