Difference between revisions of "Part:BBa K4165033"
 
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This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGS linker (BBa_K4165065), TD28rev (BBa_K4165006), GGSGGGGG linker (BBa_K4165019), WWW (BBa_K4165007), GSGS linker (BBa_K4165065), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).
 
This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGS linker (BBa_K4165065), TD28rev (BBa_K4165006), GGSGGGGG linker (BBa_K4165019), WWW (BBa_K4165007), GSGS linker (BBa_K4165065), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).
  
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===Source===
 
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Synthesized
 
  
 
===Usage and Biology===
 
===Usage and Biology===
−
Switch 13 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 PDZ peptide, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.
+
Switch 13 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 peptide binding PDZ, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 upon clamp binding to the target protein requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.
 +
 
 +
The TD28REV and WWW peptides considered as tau binding peptides are proved experimentally to bind with tau inhibit the aggregations of tau aggregations respectively. The H1A peptide was also proven to bind with the PDZ of HtrA1 experimentally. The last part is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, it will act and inhibit the Protein. The whole construction was similarly proved from literature. The process of assembly of the whole switch was done according to both CAPRI and NCBI protocols.
  
 
<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
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The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta with a score of 4 out of 6 according to our quality assessment code.
 
The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta with a score of 4 out of 6 according to our quality assessment code.
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<html>
 
<html>
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                                 Figure 1. The 3D structure of switch 13 model visualized by Pymol
 
                                 Figure 1. The 3D structure of switch 13 model visualized by Pymol
 +
 +
<h1>Switch construction Pipeline</h1>
 +
 +
<p style=" font-weight: bold; font-size:14px;"> 1) Modelling </p>
 +
<p> Since our parts do not have experimentally acquired structures, we have to model them. This approach is done using both denovo modelling (ab initio) and template-based modelling. For modelling small peptides of our system  we used AppTest and Alphafold.</p>
 +
<p style=" font-weight: bold; font-size:14px;"> 2) Structure Assessment </p>
 +
<p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link modelling page).</p>
 +
<p style=" font-weight: bold; font-size:14px;"> 3) Quality Assessment </p>
 +
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.</p>
 +
<p style=" font-weight: bold; font-size:14px;">4) Filtering</p>
 +
<p>We take the top ranked models from the previous steps.</p>
 +
<p style=" font-weight: bold; font-size:14px;">5) Docking</p>
 +
<p>The top models are docked with the protein of intereset (in our case it was the HtrA1 with a BBa_K4165004.</p>
 +
<p style=" font-weight: bold; font-size:14px;">6) Ranking</p>
 +
<p>The docking results are ranked according to their PRODIGY results. For more information: (Link Docking page).</p>
 +
<p style=" font-weight: bold; font-size:14px;">7) Top Models</p>
 +
<p>The results that came out from PRODIGY are ranked and top models are chosen to proceed with to the next step. For more information: (Link Docking page).</p>
 +
<p style=" font-weight: bold; font-size:14px;">8) Alignment</p>
 +
<p>Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex which is the HtrA1-inhibitor complex to check whether they binded to the same site or not.</p>
 +
<p style=" font-weight: bold; font-size:14px;">9) Linker length</p>
 +
<p>The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide which is between both C terminals, N terminals, C- and N- terminal, and N- and C-terminals.</p>
 +
<p style=" font-weight: bold; font-size:14px;">10) Assembly</p>
 +
<p>After settling on the linkers lengths, now we will proceed to the assembly step of the whole system which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.</p>
 +
 +
<html>
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<p>a<img src="https://static.igem.wiki/teams/4165/wiki/team-members/team-members/new/q8iub5.png" style="margin-left:50px;" alt="" width="150" />b<img src="https://static.igem.wiki/teams/4165/wiki/lh1a.png" style="margin-left:50px;" alt="" width="150" />c<img src="https://static.igem.wiki/teams/4165/wiki/team-members/team-members/new/td28rev-www.png" style="margin-left:50px;" alt="" width="150" /></p>
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</html>
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        Figure (a,b,c) : 3D structure of  Q8IUB5 Inhibitor , H1A Peptide ,  and TD28rev-GGSGGGG-WWW clamp
 +
                                    used in our assembly of switch 13
 +
 +
<p style=" font-weight: bold; font-size:14px;">11) Structure Assessment</p>
 +
<p>In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link modelling page).</p>
 +
<p style=" font-weight: bold; font-size:14px;">12) Quality Assessment </p>
 +
<p>Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.</p>
 +
 +
 +
 +
<html>
 +
<style>
 +
table, th, td {
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  border:1px solid black; margin-left:auto;margin-right:auto;
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}
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</style>
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<body>
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<table style="width:65%">
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<table>
 +
  <tr>
 +
    <th>cbeta_deviations</th>
 +
    <th>clashscore</th>
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    <th>molprobity</th>
 +
    <th>ramachandran_favored</th>
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    <th>ramachandran_outliers</th>
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    <th>Qmean_4</th>
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    <th>Qmean_6</th>
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  </tr>
 +
  <tr>
 +
    <td>0</td>
 +
    <td>5.37</td>
 +
    <td>1.46</td>
 +
    <td>97.04</td>
 +
    <td>0.74</td>
 +
    <td>0.590059</td>
 +
    <td>-0.40025</td>
 +
  </tr>
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</table>
 +
</body>
 +
</html>
 +
  
  
 +
===Conclusion===
 +
The top model was HtrA1 switch 12 (BBa_K4165032) since it was the best switch fulfilling the criteria of structure assessment, docking, and RMSD.
  
  

Latest revision as of 20:58, 13 October 2022


HtrA1 Switch number 13

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGS linker (BBa_K4165065), TD28rev (BBa_K4165006), GGSGGGGG linker (BBa_K4165019), WWW (BBa_K4165007), GSGS linker (BBa_K4165065), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).


Usage and Biology

Switch 13 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 peptide binding PDZ, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 upon clamp binding to the target protein requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.

The TD28REV and WWW peptides considered as tau binding peptides are proved experimentally to bind with tau inhibit the aggregations of tau aggregations respectively. The H1A peptide was also proven to bind with the PDZ of HtrA1 experimentally. The last part is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, it will act and inhibit the Protein. The whole construction was similarly proved from literature. The process of assembly of the whole switch was done according to both CAPRI and NCBI protocols.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 379
    Illegal AgeI site found at 115
  • 1000
    COMPATIBLE WITH RFC[1000]

Dry Lab

Modeling

The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta with a score of 4 out of 6 according to our quality assessment code. 


                               Figure 1. The 3D structure of switch 13 model visualized by Pymol

Switch construction Pipeline

1) Modelling

Since our parts do not have experimentally acquired structures, we have to model them. This approach is done using both denovo modelling (ab initio) and template-based modelling. For modelling small peptides of our system we used AppTest and Alphafold.

2) Structure Assessment

In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link modelling page).

3) Quality Assessment

Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.

4) Filtering

We take the top ranked models from the previous steps.

5) Docking

The top models are docked with the protein of intereset (in our case it was the HtrA1 with a BBa_K4165004.

6) Ranking

The docking results are ranked according to their PRODIGY results. For more information: (Link Docking page).

7) Top Models

The results that came out from PRODIGY are ranked and top models are chosen to proceed with to the next step. For more information: (Link Docking page).

8) Alignment

Docked structures are aligned. This means that the HtrA1- binding peptide complex is aligned with the second complex which is the HtrA1-inhibitor complex to check whether they binded to the same site or not.

9) Linker length

The linker lengths are acquired by seeing the distance between the inhibitor and the HtrA1 binding peptide which is between both C terminals, N terminals, C- and N- terminal, and N- and C-terminals.

10) Assembly

After settling on the linkers lengths, now we will proceed to the assembly step of the whole system which is done using TRrosetta, AlphaFold, RosettaFold, and Modeller.

abc

        Figure (a,b,c) : 3D structure of  Q8IUB5 Inhibitor , H1A Peptide ,  and TD28rev-GGSGGGG-WWW clamp 
                                    used in our assembly of switch 13

11) Structure Assessment

In order to assess the quality of our structures we used the Swiss-Model tool which gives an overall on quality of any 3D structure (For more information: (Link modelling page).

12) Quality Assessment

Using the code created by us (CU_Egypt 2022), we use the JSON files created from the structure assessment step in Swiss-Model to rank all the models For more information: (Link software page) under the name of Modric.


cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 5.37 1.46 97.04 0.74 0.590059 -0.40025


Conclusion

The top model was HtrA1 switch 12 (BBa_K4165032) since it was the best switch fulfilling the criteria of structure assessment, docking, and RMSD.