Difference between revisions of "ABC-INT-Homology modelling"

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You need to complete the following units before beginning this one:
 
You need to complete the following units before beginning this one:
 
*[[BIN-SX-Homology_modelling|BIN-SX-Homology_modelling (Homology Modeling)]]
 
*[[BIN-SX-Homology_modelling|BIN-SX-Homology_modelling (Homology Modeling)]]
−
 
−
{{Vspace}}
 
−
 
−
 
−
=== Objectives ===
 
−
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "objectives" -->
 
−
...
 
−
 
−
{{Vspace}}
 
−
 
−
 
−
=== Outcomes ===
 
−
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "outcomes" -->
 
−
...
 
  
 
{{Vspace}}
 
{{Vspace}}
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=== Deliverables ===
 
=== Deliverables ===
 
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "deliverables" -->
 
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "deliverables" -->
−
<!-- included from "ABC-unit_components.wtxt", section: "deliverables-milestone" -->
+
<!-- included from "ABC-unit_components.wtxt", section: "deliverables-integrator" -->
−
*<b>No separate deliverables</b>: This unit collects other units and has no deliverables on its own.
+
*<b>Integrator unit</b>: Deliverables will be marked as detailed on this page.
  
 
{{Vspace}}
 
{{Vspace}}
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=== Evaluation ===
 
=== Evaluation ===
 
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "evaluation" -->
 
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "evaluation" -->
−
<!-- included from "ABC-unit_components.wtxt", section: "eval-INT-TBD" -->
+
;This "Integrator Unit" should be submitted for evaluation for a maximum of 10 marks.
−
<b>Evaluation: Integrated Unit</b><br />
+
:Please note the evaluation types that are available as options for this unit. Choose one evaluation type that you have not chosen for another Integrator Unit. (Each submitted Integrator Unit must be evaluated in a different way and one of your evaluations - but not your first one - must be an oral exam).
−
:This unit should be submitted for evaluation for a maximum of 10 marks. Details TBD.
+
{{Smallvspace}}
 +
;Report option
 +
* Work through the tasks described in the scenario.
 +
* Document your results in a short report on a subpage of your User page on the Student Wiki. Describe your methods (R-code!) in an appendix;
 +
* When you are done with everything, add the following category tag to the page:
 +
::<code><nowiki>[[Category:EVAL-INT-Homology_modelling]]</nowiki></code>
 +
:'''Do not''' change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 +
{{Smallvspace}}
 +
;Figure option
 +
* Work through the tasks described in the scenario.
 +
* Document your results in a short report on a subpage of your User page on the Student Wiki. Describe your methods (R-code!) in an appendix;
 +
* When you are done with everything, add the following category tag to the page:
 +
::<code><nowiki>[[Category:EVAL-INT-Homology_modelling]]</nowiki></code>
 +
:'''Do not''' change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 +
{{Smallvspace}}
 +
;Interview option
 +
: Identify a laboratory whose work has recently included producing and interpreting a homology model. Get in touch with the PI, a postdoc or senior graduate student in the laboratory and interview them in person or by eMail. Find out
 +
* why this work is important;
 +
* how they approach it methodologically;
 +
* in particular, how they interpret the model and what the model tells them taht a sequence alignment alone would not have;
 +
* what they have recently learned.
 +
* write up your interview on a subpage of your User page of the Student Wiki;
 +
* add information that may be required to understand the context;
 +
* make sure that you included important literature references.
 +
* Make sure your interviewee is aware of what this is for, has given permission to use your interview for course credit and perhaps to augment the learning unit.
 +
* Make sure contact information for your interviewee is included on your page.
 +
* Add a CC-BY tag to your submission.
 +
*When you are done with everything, add the following category tag to the page:
 +
::<code><nowiki>[[Category:EVAL-INT-Homology_modelling]]</nowiki></code>
 +
:'''Do not''' change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 +
<!--
 +
{{Smallvspace}}
 +
;Literature research option
 +
: ...
 +
-->
 +
{{Smallvspace}}
 +
;Oral exam option
 +
* Work through the tasks described in the scenario. Remember to document your work in your journal.
 +
* Part of your task will involve writing an R script, place that code in a subpage of your User page on the Student Wiki and link to it from your Journal. (Do not add an evaluation category tag to that code).
 +
* Your work must be complete before 21:00 on the day of your exam.
 +
* Schedule an oral exam by editing the [http://steipe.biochemistry.utoronto.ca/abc/students/index.php/Signup-Oral_exams_2017 '''signup page on the Student Wiki''']. Enter the unit that you are signing up for, and your name. You must have signed-up for an exam slot before 21:00 on the day before your exam.
 +
{{Smallvspace}}
 +
;R code option
 +
* Work through the tasks described in the scenario and develop code as required.
 +
* Put your code on a subpage of your User page on the Student Wiki;
 +
* When you are done with everything, add the following category tag to the page:
 +
::<code><nowiki>[[Category:EVAL-INT-Homology_modelling]]</nowiki></code>
 +
:'''Do not''' change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 +
 
  
 
{{Vspace}}
 
{{Vspace}}
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== Contents ==
 
== Contents ==
 
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "contents" -->
 
<!-- included from "../components/ABC-INT-Homology_modelling.components.wtxt", section: "contents" -->
−
<!-- included from "ABC-unit_components.wtxt", section: "milestone" -->
 
−
This is a "milestone unit". Its purpose is merely to collect a number of preparatory units into a single, common prerequisite. It has no contents of its own; you are expected to be familiar and competent with all preparatory material at this point.
 
  
 +
{{Vspace}}
  
−
=== The DNA binding site ===
+
=== Scenario background ===
  
 +
You have collected the APSES domain proteins of MYSPE in your protein database. This collection of proteins now contains orthologues and paralogues. It is reasonable to assume that orthologues conserve structure and function, whereas paralogues conserve structure, but change function - in particular, paralogous APSES domains would be expected to recognize different DNA binding sites.
  
−
Now, that you know how MYSPE Mbp1 aligns with yeast Mbp1, you can evaluate functional conservation in these homologous proteins. You probably already downloaded the two Biochemistry papers by Taylor et al. (2000) and by Deleeuw et al. (2008) that we encountered in Assignment 2. These discuss the residues involved in DNA binding<ref>([http://www.ncbi.nlm.nih.gov/pubmed/10747782 Taylor ''et al.'' (2000) ''Biochemistry'' '''39''': 3943-3954] and [http://www.ncbi.nlm.nih.gov/pubmed/18491920 Deleeuw ''et al.'' (2008) Biochemistry. '''47''':6378-6385])</ref>. In particular the residues between 50-74 have been proposed to comprise the DNA recognition domain.
 
  
 +
 +
=== For the Report Option ...===
 
{{task|
 
{{task|
−
# Using the APSES domain alignment you have just constructed, find the MYSPE Mbp1 residues that correspond to the range 50-74 in yeast.
+
# Produce two separate MSAs, one for the Mbp1 orthologues, and one for the other APSES domain containing sequences in <code>myDB</code>. Save the MSA for the APSES domain only as a multi FASTA file that can be read by Chimera.
−
# Note whether the sequences are especially highly conserved in this region.
+
# In Chimera, create a model of your MBP1_MYSPE APSES domain bound to DNA, based on the <code>4UX5</code> structure - (Superimpose your homology model on Chain A and delete Chain B).
−
# Using Chimera, look at the region. Use the sequence window '''to make sure''' that the sequence numbering between the paper and the PDB file are the same (they are often not identical!). Then select the residues - the proposed recognition domain -  and color them differently for emphasis. Study this in stereo to get a sense of the spatial relationships. Check where the conserved residues are.
+
# Create a copy of that model.
−
# A good representation is '''stick''' - but other representations that include sidechains will also serve well.
+
# Colour the original by conservation scores of the Mbp1 APSES domain MSA, and colour the copy with the conservation scores for the other APSES domains.
−
# Calculate a solvent accessible surface of the protein in a separate representation and make it transparent.
+
# Identify residues that appear functionally conserved - i.e. potentially contributing to DNA binding specificity; conserved in Mbp1 orthologues, but variable in the other domains.
−
# You could  combine three representations: (1) the backbone (in '''ribbon view'''), (2) the sidechains of residues that presumably contact DNA, distinctly colored, and (3) a transparent surface of the entire protein. This image should show whether residues annotated as DNA binding form a contiguous binding interface.
+
# Identify residues that are structurally conserved, i.e. conserved in all APSES domains.
 +
# Illustrate your findings with stereo images and write a brief technical report. Make sure that your description is specific with respect to actually identifying sequence numbers and residue types.
 
}}
 
}}
  
 +
{{Vspace}}
  
−
DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone.
+
=== For the Oral Exam Option ...===
 +
{{task|1=
  
 +
What can be learned from the two different binding modes of chains A and B of 4UX5, regarding the APSES domain of MBP1_MYSPE?
  
 +
# In Chimera, create a model of your MBP1_MYSPE APSES domain bound to DNA, based on the <code>4UX5</code> structure - (Superimpose your homology model on 4UX5 Chain A).
 +
# Create a copy of 4UX5 Chain B and superimpose that too on Chain A.
 +
# Colour all protein and DNA chains by element. Then colour the C-atoms of your model and the two 4UX5 chains with distinct colours that can't be confused with N, O, S, and P atoms. This makes it possible to identify different chains, while still studying details of interactions. Select all residues that are within 5.0 Å of DNA and display them as sticks. Display the rest of the protein as ribbons or tubes. Display the water molecules too.
 +
# Set the pivot to a residue at the protein DNA interface.
 +
# Use '''Save Session''' to save the entire scene to file.
 +
# Be prepared to reload the scene on your laptop during the oral exam and explain how the findings in 4UX5 relate to your model.
 +
 +
}}
 +
 +
{{Vspace}}
 +
 +
=== For the Publication Image Option ...===
 
{{task|
 
{{task|
−
*Study and consider whether this is the case here and which residues might be included.
+
 
 +
DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone. Of course, your homology model did not take the DNA ligand into account.
 +
 
 +
# In Chimera, create a model of your MBP1_MYSPE APSES domain bound to DNA, based on the <code>4UX5</code> structure - (Superimpose your homology model on Chain A and delete Chain B).
 +
# Create a publication quality image (wall-eyed stereo) with two panels: (a) shows the conserved positively charged residues of MBP1_MYSPE that bind to DNA (labels!) in context of the bound DNA, (b) shows the solvent excluded surface calculated separately for protein and DNA, colored by Coulomic surface coloring. Make the surface sufficiently transparent to show the underlying ribbon representations of the backbone, and the side-chains of the conserved positively charged residues. Your Figure (A) is probably best done as a stick or sphere model. Figure (B) may combine (i) the protein and DNA backbones (in '''ribbon view'''), (ii) the sidechains of residues that your are discussing, distinctly coloured, (iii) a transparent surface of the protein, and (iv) a tranparent surface of the DNA. The goal is to demonstrate that the residue conservation of positively charged residues can be explained by their contribution to a surface that is electrostatically complementary to DNA. Make sure your figure does not include irrelevant items that obscure the message.
 +
# Write an explicit, descriptive figure caption.
 +
# Print your figure and figure caption as a PDF and upload to the Student Wiki.
 +
# On your submission page, describe the steps that you went through to create the images and link to your PDF.
 
}}
 
}}
  
 +
{{Vspace}}
 +
 +
=== For the R-code option ...===
 +
{{task|
 +
 +
How different are homology models based on 1BM8 and 4UX5? Where are the important differences?
  
 +
# Produce two homology models for MBP1_MYSPE: one with the 1BM8 template, the other with the 4UX5 template (You already have one of the two).
 +
# Write an R script using bio3d to superimpose the two models, calculate the RMSD between each residue pair, writes the RMSD to each residues' B Factor field for both of the two models and save the resulting PDB files.
 +
# In Chimera, load the two models, superimpose them, and color them by the RMSD values you have computed.
 +
# Save a stereo image.
 +
# Submit your script and the image. Remember to comment your script.
  
 +
}}
  
 +
{{Vspace}}
  
  
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:2017-08-05
 
:2017-08-05
 
<b>Modified:</b><br />
 
<b>Modified:</b><br />
−
:2017-08-09
+
:2017-10-31
 
<b>Version:</b><br />
 
<b>Version:</b><br />
−
:0.1
+
:1.0
 
<b>Version history:</b><br />
 
<b>Version history:</b><br />
 +
*1.0 Live 2017
 
*0.1 First stub
 
*0.1 First stub
 
</div>
 
</div>

Revision as of 22:49, 31 October 2017

Integration Unit: Homology Modelling


 

Keywords:  Integration unit: create a homology model and assess the role of sequence conservation


 



 


Sorry!

This page is only a stub; it is here as a placeholder to establish the logical framework of the site but there is no significant content as yet. Do not work with this material until it is updated to "live" status.


 


Abstract

This page assesses the learning units for working with multiple sequence alignments and structure data.

  • Use an Mbp1 orthologue (RBM)
  • Find all orthologues in MYSPE and reference species
  • Homology model
  • Calculate conservation scores
  • Map, visualize
  • Interpret with reference to winged HTH domains: 1VTN (canonical), 4UX5 (M. Oryzae Mbp1), 1DP7, 3HTS.


 


This unit ...

Prerequisites

You need to complete the following units before beginning this one:


 


Deliverables

  • Integrator unit: Deliverables will be marked as detailed on this page.


 


Evaluation

This "Integrator Unit" should be submitted for evaluation for a maximum of 10 marks.
Please note the evaluation types that are available as options for this unit. Choose one evaluation type that you have not chosen for another Integrator Unit. (Each submitted Integrator Unit must be evaluated in a different way and one of your evaluations - but not your first one - must be an oral exam).
 
Report option
  • Work through the tasks described in the scenario.
  • Document your results in a short report on a subpage of your User page on the Student Wiki. Describe your methods (R-code!) in an appendix;
  • When you are done with everything, add the following category tag to the page:
[[Category:EVAL-INT-Homology_modelling]]
Do not change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 
Figure option
  • Work through the tasks described in the scenario.
  • Document your results in a short report on a subpage of your User page on the Student Wiki. Describe your methods (R-code!) in an appendix;
  • When you are done with everything, add the following category tag to the page:
[[Category:EVAL-INT-Homology_modelling]]
Do not change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 
Interview option
Identify a laboratory whose work has recently included producing and interpreting a homology model. Get in touch with the PI, a postdoc or senior graduate student in the laboratory and interview them in person or by eMail. Find out
  • why this work is important;
  • how they approach it methodologically;
  • in particular, how they interpret the model and what the model tells them taht a sequence alignment alone would not have;
  • what they have recently learned.
  • write up your interview on a subpage of your User page of the Student Wiki;
  • add information that may be required to understand the context;
  • make sure that you included important literature references.
  • Make sure your interviewee is aware of what this is for, has given permission to use your interview for course credit and perhaps to augment the learning unit.
  • Make sure contact information for your interviewee is included on your page.
  • Add a CC-BY tag to your submission.
  • When you are done with everything, add the following category tag to the page:
[[Category:EVAL-INT-Homology_modelling]]
Do not change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.
 
Oral exam option
  • Work through the tasks described in the scenario. Remember to document your work in your journal.
  • Part of your task will involve writing an R script, place that code in a subpage of your User page on the Student Wiki and link to it from your Journal. (Do not add an evaluation category tag to that code).
  • Your work must be complete before 21:00 on the day of your exam.
  • Schedule an oral exam by editing the signup page on the Student Wiki. Enter the unit that you are signing up for, and your name. You must have signed-up for an exam slot before 21:00 on the day before your exam.
 
R code option
  • Work through the tasks described in the scenario and develop code as required.
  • Put your code on a subpage of your User page on the Student Wiki;
  • When you are done with everything, add the following category tag to the page:
[[Category:EVAL-INT-Homology_modelling]]
Do not change your submission page after this tag has been added. The page will be marked and the category tag will be removed by the instructor.


 


Contents

 

Scenario background

You have collected the APSES domain proteins of MYSPE in your protein database. This collection of proteins now contains orthologues and paralogues. It is reasonable to assume that orthologues conserve structure and function, whereas paralogues conserve structure, but change function - in particular, paralogous APSES domains would be expected to recognize different DNA binding sites.


For the Report Option ...

Task:

  1. Produce two separate MSAs, one for the Mbp1 orthologues, and one for the other APSES domain containing sequences in myDB. Save the MSA for the APSES domain only as a multi FASTA file that can be read by Chimera.
  2. In Chimera, create a model of your MBP1_MYSPE APSES domain bound to DNA, based on the 4UX5 structure - (Superimpose your homology model on Chain A and delete Chain B).
  3. Create a copy of that model.
  4. Colour the original by conservation scores of the Mbp1 APSES domain MSA, and colour the copy with the conservation scores for the other APSES domains.
  5. Identify residues that appear functionally conserved - i.e. potentially contributing to DNA binding specificity; conserved in Mbp1 orthologues, but variable in the other domains.
  6. Identify residues that are structurally conserved, i.e. conserved in all APSES domains.
  7. Illustrate your findings with stereo images and write a brief technical report. Make sure that your description is specific with respect to actually identifying sequence numbers and residue types.


 

For the Oral Exam Option ...

Task:
What can be learned from the two different binding modes of chains A and B of 4UX5, regarding the APSES domain of MBP1_MYSPE?

  1. In Chimera, create a model of your MBP1_MYSPE APSES domain bound to DNA, based on the 4UX5 structure - (Superimpose your homology model on 4UX5 Chain A).
  2. Create a copy of 4UX5 Chain B and superimpose that too on Chain A.
  3. Colour all protein and DNA chains by element. Then colour the C-atoms of your model and the two 4UX5 chains with distinct colours that can't be confused with N, O, S, and P atoms. This makes it possible to identify different chains, while still studying details of interactions. Select all residues that are within 5.0 Å of DNA and display them as sticks. Display the rest of the protein as ribbons or tubes. Display the water molecules too.
  4. Set the pivot to a residue at the protein DNA interface.
  5. Use Save Session to save the entire scene to file.
  6. Be prepared to reload the scene on your laptop during the oral exam and explain how the findings in 4UX5 relate to your model.


 

For the Publication Image Option ...

Task:


DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone. Of course, your homology model did not take the DNA ligand into account.

  1. In Chimera, create a model of your MBP1_MYSPE APSES domain bound to DNA, based on the 4UX5 structure - (Superimpose your homology model on Chain A and delete Chain B).
  2. Create a publication quality image (wall-eyed stereo) with two panels: (a) shows the conserved positively charged residues of MBP1_MYSPE that bind to DNA (labels!) in context of the bound DNA, (b) shows the solvent excluded surface calculated separately for protein and DNA, colored by Coulomic surface coloring. Make the surface sufficiently transparent to show the underlying ribbon representations of the backbone, and the side-chains of the conserved positively charged residues. Your Figure (A) is probably best done as a stick or sphere model. Figure (B) may combine (i) the protein and DNA backbones (in ribbon view), (ii) the sidechains of residues that your are discussing, distinctly coloured, (iii) a transparent surface of the protein, and (iv) a tranparent surface of the DNA. The goal is to demonstrate that the residue conservation of positively charged residues can be explained by their contribution to a surface that is electrostatically complementary to DNA. Make sure your figure does not include irrelevant items that obscure the message.
  3. Write an explicit, descriptive figure caption.
  4. Print your figure and figure caption as a PDF and upload to the Student Wiki.
  5. On your submission page, describe the steps that you went through to create the images and link to your PDF.


 

For the R-code option ...

Task:


How different are homology models based on 1BM8 and 4UX5? Where are the important differences?

  1. Produce two homology models for MBP1_MYSPE: one with the 1BM8 template, the other with the 4UX5 template (You already have one of the two).
  2. Write an R script using bio3d to superimpose the two models, calculate the RMSD between each residue pair, writes the RMSD to each residues' B Factor field for both of the two models and save the resulting PDB files.
  3. In Chimera, load the two models, superimpose them, and color them by the RMSD values you have computed.
  4. Save a stereo image.
  5. Submit your script and the image. Remember to comment your script.



 


 


Further reading, links and resources

 


Notes


 


Self-evaluation

 



 



 

If in doubt, ask! If anything about this learning unit is not clear to you, do not proceed blindly but ask for clarification. Post your question on the course mailing list: others are likely to have similar problems. Or send an email to your instructor.


 

About ...
 
Author:

Boris Steipe <boris.steipe@utoronto.ca>

Created:

2017-08-05

Modified:

2017-10-31

Version:

1.0

Version history:

  • 1.0 Live 2017
  • 0.1 First stub

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