User:Boris/Temp/APB

Note! This assignment is currently active. All significant changes will be announced on the mailing list.

 
 

   

   

Assignment 2 - Search, retrieve and annotate

   

Preparation, submission and due date

Read carefully.

Be sure you have understood all parts of the assignment and cover all questions in your answers! Sadly, we always get assignments back in which important aspects have simply been overlooked and marks are unnecessarily lost. Sadly, we always get assignments back in which important aspects have simply been overlooked and marks are unnecessarily lost. If you did not notice that the above sentence was repeated, you are not reading carefully enough.

Review the guidelines for preparation and submission of BCH441 assignments.

The due date for the assignment is Thursday, October 24 at 12:00 noon (before the quiz).

   

Your documentation for the procedures you follow in this assignment will be worth 1 mark.

   

Introduction

Baker's yeast, Saccharomyces cerevisiae, is perhaps the most important model organism. It is a eukaryote that has been studied genetically and biochemically in great detail for many decades, and it is easily manipulated with high-throughput experimental methods. We will use information from this model organism to study the conservation of function and sequence in other fungi whose genomes have been completely sequenced; the assignments are an exercise in model-organism reasoning: the transfer of knowledge from one, well-studied organism to others.

This and the following assignments will revolve around a transcription factor that plays an important role in the regulation of the cell cycle: Mbp1 is a key component of the MBF complex (Mbp1/Swi6). This complex regulates gene expression at the crucial G1/S-phase transition of the mitotic cell cycle and has been shown to bind to the regulatory regions of more than a hundred target genes.

One would speculate that such central control machinery would be conserved in other fungi and it will be your task in these assignments to collect evidence whether related molecular machinery is present in some of the newly sequenced fungal genomes. Throughout the assignments we will use freely available tools to conduct bioinformatics investigations of questions such as:

• What functional features can we detect in Mbp1?
• Do homologous proteins exist in other organisms?
• Do we believe these homologues may bind to similar sequence motifs?
• Do we believe they may function in a similar way?
• Do other organisms appear to have related cell-cycle control systems?

 

• Access the information page on Mbp1 at the Saccharomyces Genome Database and read the summary paragraph on the protein's function!

(If you would like to brush up on the concepts mentioned above, you could study the corresponding chapter in Lodish's Molecular Cell Biology and./or read Nobel laureate Paul Nurse's review (pdf) of the key concepts of the eukaryotic cycle. It is not strictly necessary to understand the details of the yeast cell-cycle to complete the assignments, but recommended, since it's obviously more fun to work with concepts that actually make some sense.)

In this particular assignment you will go on a search and retrieve mission for information on yeast Mbp1, using common public databases and Web resources.

Retrieve

Much useful information on yeast Mbp1 is compiled at the SGD information page on Mbp1. However we don't always have the luxury of such precompiled information. Let's look at the protein and it's features "the traditional way".

• Navigate to the NCBI homepage (you probably have bookmarked it anyway) and enter Mbp1 AND "saccharomyces cerevisiae"[organism] as an Entrez query.
• Click on Protein and find the RefSeq record for the protein sequence.
• From the NCBI RefSeq record, obtain a FASTA sequence of the protein and paste it into your assignment.

There are several sources for functional domain annotations of proteins. The NCBI has the Conserved Domain Database, in Europe, the SMART database provides such annotations. In terms of domains, both resources are very comparable. But SMART also analyses more general features such as low-complexity sequences and coiled coils. In order to use SMART however, we need the Uniprot accession number that corresponds to the refseq identifier. In a rational world, one would wish that such important crossreferences would simply be provided by the NCBI ... well, we have been wishing this for many years now. Fortunately ID-mapping services exist.

• Navigate to the UniProt ID-Mapping service. Enter the RefSeq identifier for the yeast Mbp1 protein and retrieve the corresponding UniProtKB Accession number. If this does not work, try the same mapping at the PIR ID-mapping service. Note the Uniprot accession number you find. (Should this work equally on both sites?)

Now navigate to Uniprot, enter the ID you have found into the search field and select [Sequence Clusters(UniRef)] as the database to search in. There should be two sequences in the [UniRef100 ... (100% identical)] cluster. Compare them. One of them is a highly annotated Swiss-Prot record, the other is practically unannotated data that has been imported from a "third party" to UniProt. Unfortunately, that one is the sequence that the ID mapping service had found. No cross-references to the NCBI are included with Swiss-Prot records, nor do NCBI RefSeq records cross-reference NCBI holding. I consider this a sorry state of affairs. Therefore most of us actually run BLAST searches to find equivalent sequences in other databases and this is the most wasteful way imaginable to address the problem.

• Note down the SwissProt ID and the UniProtKB Accession Number for yeast Mbp1.

Analyse

   

saccharomyces cerevisiae Mbp1 - domain annotations

Now we can analyse Mbp1's domain in SMART, and use this information to annotate the sequence in detail.

• Navigate to the SMART database, use "Normal Mode", then in the "Sequence Analysis" form enter the UniProtKB yeast Mbp1 accession number, check the checkboxes for the aditional analyses that SMART offers and carefully review the results. By that I mean I might ask at some point what a particular section of the result means and how it is interpreted re. its biological significance. If parts are not obvious to you: → mailing list.
• In your assignment, in the actual, full-length sequence, highlight or otherwise clearly identify the features that SMART has annotated. Minimally you should include KilA-N, low complexity, coils, and Ankyrin domains, taking the sequence coordinates from the SMART annotations. Make sure you highlight the whole length of the feature and get the boundaries right. If features overlap, you could eg. highlight one red, the other green and the overlap yellow. Or underline one, format the other in italics... And don't forget to label 'what you have highlighted.

APSES (KilA-N) domains

As you see from the annotations, Mbp1 is a large protein comprising several domains; it binds DNA through a small domain called the APSES domain (this is reported as the KilA-N domain superfamily). Many organisms have transcription factors that have a domains homologous to other APSES domains. Since we are interested in related proteins, and all functional relatives would be expected to share such a DNA binding domain, we should define this domain in more detail in order to be able to use it later to search for homologous proteins in diverse organisms.  

Use the NCBI Entrez system to search for the string "apses" in the "Conserved Domains" database and access the entry for the KilA-N domain superfamily. You should find 10 aligned sequences on that page, each with their own GI identifier. To find the actual boundaries of the domain annotation, do the following:

• Navigate back to the RefSeq Protein record (You did record that link in your documentation, right?). In the right-hand menu, find the section "Related information" and click on CDD Search results.
• Click on the colored box for one of the annotated domains to appreciate the level of detailedinformation thatnis available here.
• Back in the CDD annotation window, click on the [+] next to KilA-N super family to access the actual alignment of the PFAM domain definition with the MBP1 sequence.
• Check and record (e.g. by highlighting) whether the NCBI and the SMART definition of the APSES domain in Mbp1 coincide exactly. If they don't, explain briefly what that means.
• Make sure you understand how the sequences displayed on the CDD page and the actual domain sequences differ. Hint: not all sequences are displayed in their full-length.

APSES domain structure

We can expect that the structures of all homologous APSES domains should be similar, i.e. if the structure of one is known, we should be able to conclude the approximate three-dimensional structure of any APSES domain. Indeed, structural information is available for APSES domains!

There are several possible approaches you could pursue to identify candidate files:

• there may be cross references to structures/PDB on any of the pages you have visited;
• you could search the PDB itself for the keyword Mbp1;
• you could use the domain sequence you have defined and ;
  • BLAST it against the database of PDB sequences (select it as an option on the BLAST form);
  • perform an "advanced serach" for similar sequences on the PDB Website.

In any case, you should find more than one coordinate files that contain MBP1-like structures. Therefore you need to make a choice which one is the "best" for further analysis. Your choice of the "best" file for study could be based on:

• experimental method (X-ray or NMR);
• quality of the structure (resolution, refinement);
• size (coverage) of the structure (number of amino acids for which structure coordinates have been determined).

 

 

• Record how you have identified the file you consider the "best" and what criteria you have used to define whether it is better suited for analysis than others.

DNA binding site

The Mbp1 APSES domain has been shown to bind to DNA and the residues involved in DNA binding have been characterized. (Taylor et al. (2000) Biochemistry 39: 3943-3954 and Deleeuw et al. (2008) Biochemistry. 47:6378-6385) . In particular the residues between 50-74 have been proposed to comprise the DNA recognition domain.

Analysis (0.5 marks)

• Using VMD, generate a parallel stereo view of the protein structure that clearly shows the proposed Mbp1 DNA recognition domain, distinctly coloured differently from the rest of the protein. Use a representation that includes the sidechains.

• Generate a second VMD stereo image as above, but use a representation that emphasizes the secondary structure of the structure (tube or cartoon representation, colouring by structure).

• Generate a third VMD stereo image that shows three representations combined: (1) the backbone, (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. Note: VMD makes smart use of GPU capabilities of your computer. Try setting your graphics parameters to visualize with GLSL - your transparent surface may look much better.

Include the images into your assignment but be careful not to exceed the width and size restrictions I have defined in the submission guidelines. Include your "selections statements" (e.g. protein and not resid 23 to 34) so that it is easy for you to reproduce what you have done. Also note any important parameters you have changed from the default.

DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone.

Analysis (1 mark)

• Report whether this is the case here and which residues might be included.
• Do the DNA binding residues form a contiguous surface that is compatible with a binding interface? Justify your conclusions.

Be specific in your analysis: write exactly which residue does what. For example don't write

there are many lysines...

but write something like

K33, R35 and K76 form a patch of positively charged residues close to the C-terminus of the putative recognition helix.

This is the interpretation of results and therefore the most important step of your entire analysis.

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== More statistics with R== 

Time for a break:

Second step (0.5 marks)

Access the Clarkson University R tutorial. Work through part two of the tutorial (Data types).

[End of assignment]

If you have any questions at all, don't hesitate to mail me at boris.steipe@utoronto.ca or post your question to the Course Mailing List