ABC-INT-Homology modelling

Integrator Unit: Homology Modelling

(Integrator unit: create a homology model and assess the role of sequence conservation)

 

Abstract:

This page integrates material from the learning units for working with multiple sequence alignments and structure data in a task for evaluation.

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Deliverables:

Integrator unit: Deliverables can be submitted for course marks. See below for details.

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Prerequisites:
This unit builds on material covered in the following prerequisite units:

• BIN-SX-Homology_modelling (Homology Modeling)

 

Please hold!

This page still needs to undergo revisions. Do not work on these tasks yet, and do not prepare contents for submission.

 

 

Evaluation

This "Integrator Unit" should be submitted for evaluation for a maximum of 8 marks if one of the written deliverables is chosen, resp. 16 marks for the oral test.

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 test).

 

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 end of page:

[[Category:EVAL-INT-Homology_modelling]].

Once the page has been saved with this tag, it is considered "submitted". Do not change your submission after this tag has been added. The page will be marked and the category tag will be removed by the instructor.

 

Publication Image 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 end of page:

[[Category:EVAL-INT-Homology_modelling]].

Once the page has been saved with this tag, it is considered "submitted". Do not change your submission after this tag has been added. The page will be marked and the category tag will be removed by the instructor.

 

Oral test option

• Work through the tasks described in the scenario. Remember to document your work in your journal.
• Your work must be complete before 21:00 on the day before your exam.
• Schedule an oral test 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 end of page:

[[Category:EVAL-INT-Homology_modelling]].

Once the page has been saved with this tag, it is considered "submitted". Do not change your submission 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.

- Model Ankyrin domain - Compute conservation scores - color residues by conservation score - Do conserved residues patch?

- Report

- R-code

 - quantify this by computing sasa
 - color surface by conservation score
 - compute conservation as a function of sasa and intramolecular contacts
 - distinguish structurally and functionally conserved residues

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; we expect them to be conserved in Mbp1 orthologues, but variable in the other sequences.
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 Test 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 test 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 Coulombic 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 but it's your figure so channel your creative talent for information design. 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. Don't forget to comment your script.

 

Self-evaluation

Further reading, links and resources

Notes

 

About ...
 
Author:

Boris Steipe <boris.steipe@utoronto.ca>

Created:

2017-08-05

Modified:

2017-10-31

Version:

1.1

Version history:

• 1.1 Corrected posted marks, which were not consistent with the description in the syllabus.
• 1.0 Live 2017
• 0.1 First stub

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Please hold!

This page still needs to undergo revisions. Do not work on these tasks yet, and do not prepare contents for submission.