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| − | Assignment 4 - Homology modeling | + | Assignment 2 - Search, retrieve and annotate |
| | </div> | | </div> |
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| − | <div style="padding: 15px; background: #F0F1F7; border:solid 1px #AAAAAA; font-size:125%;color:#444444">
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| − | ;How could the search for ultimate truth have revealed so hideous and visceral-looking an object?
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| − | ::''<small>Max Perutz (on his first glimpse of the Hemoglobin structure)</small>''
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| − | </div>
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| − | Where is the hidden beauty in structure, and where, the "ultimate truth"? In the previous assignments we have studied sequence conservation in APSES family domains and we have discovered homologues in all fungal species. This is an ancient protein family that had already duplicated to several paralogues at the time the cenancestor of all fungi lived, more than 600,000,000 years ago, in the [http://www.ucmp.berkeley.edu/fungi/fungifr.html Vendian period] of the Proterozoic era of Precambrian times.
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| − | In order to understand how specific residues in the sequence contribute to the putative function of the protein, and why and how they are conserved throughout evolution, we would need to study an explicit molecular model of an APSES domain protein, bound to its cognate DNA sequence. Explanations of a protein's observed properties and functions can't rely on the general fact that it binds DNA, we need to consider details in terms of specific residues and their spatial arrangement. In particular, it would be interesting to correlate the conservation patterns of key residues with their potential to make specific DNA binding interactions. Unfortunately, no APSES domain structures in complex with bound DNA has been solved up to now, and the experimental evidence we have considered in Assignment 2 ([http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10747782 Taylor ''et al.'', 2000]) is not sufficient to unambiguously define the details of how a DNA double helix might be bound. Moreover, at least two distinct modes of DNA binding are known for proteins of the winged-helix superfamily, of which the APSES domain is a member.
| + | {{Template:Preparation| |
| | + | care=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.| |
| | + | num=2| |
| | + | ord=second| |
| | + | due = Thursday, October 9. at 10:00 in the morning}} |
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| − | ''In this assignment you will (1) construct a molecular model of the Mbp1 orthologue in your assigned organism, (2) identify similar structures of distantly related domains for which protein-DNA complexes are known, (3) assemble a hypothetical complex structure and(4) discuss whether the available evidence allows you to distinguish between different modes of ligand binding, ''
| + | ;Your documentation for the procedures you follow in this assignment will be worth 1 mark. |
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| − | For the following, please remember the following terminology:
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| − | ;Target | + | |
| − | :The protein that you are planning to model.
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| − | ;Template
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| − | :The protein whose structure you are using as a guide to build the model.
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| − | ;Model
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| − | :The structure that results from the modeling process. It has the '''Target sequence''' and is similar to the '''Template structure'''.
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| − | A brief overview article on the construction and use of homology models is linked to the resource section at the bottom of this page. That section also contains links to other sites and resources you might find useful or interesting.
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| − | {{Template:Preparation|
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| − | care=Be sure you have understood all parts of the assignment and cover all questions in your answers! Sadly, we see too many assignments which, arduously effected, nevertheless intimate nescience of elementary tenets of molecular biology. If the sentence above did not trigger an urge to open a dictionary, you are trying to guess, rather than confirm possibly important information.|
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| − | num=4|
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| − | ord=fourth|
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| − | due = Monday, November 12 at 10:00 in the morning}}
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| − | | + | <div style="padding: 2px; background: #F0F1F7; border:solid 1px #AAAAAA; font-size:125%;color:#444444"> |
| − | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;"> | + | Introduction |
| − | ==(1) Preparation==
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| | </div> | | </div> |
| | + | Baker's yeast, ''Saccharomyces cerevisiae'', is perhaps the most important [http://en.wikipedia.org/wiki/Model_organism 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. |
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| | + | 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. |
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| − | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;">
| + | 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: |
| − | ===(1.1) Template choice and sequence (1 mark)===
| + | *What functional features can we detect in Mbp1? |
| − | </div>
| + | *Do homologous proteins exist in other organisms? |
| − | <br>
| + | *Do we believe these homologues may bind to similar sequence motifs? |
| − | Often more than one related structure can be found in the PDB. We have touched on principles of selecting template structures in the lecture and there is a short summary of [[Template_choice_principles|template choice principles]] on this Wiki. One can either search the PDB itself through its '''Advanced Search''' interface; for example one can search for sequence similarity with a BLAST search, or search for structural similarity by accessing structures according to their CATH or SCOP classification. But one can always also use the BLAST interface at the NCBI, since the sequences contained in PDB files are accessible as a database subsection on the BLAST menu.
| + | *Do we believe they may function in a similar way? |
| − | | + | *Do other organisms appear to have related cell-cycle control systems? |
| − | <div style="padding: 5px; background: #DDDDEE;">
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| − | *Use the NCBI BLAST interface to identify all PDB files that are clearly homologous to your target APSES domain, if you haven't already done so in Assignment 2. Document that you have searched in the correct subsection of the database by selecting "pdb" on the database options menu. For the hits you find, consider how these coordinate sets differ and which features would make each more or less suitable for your task by commenting briefly on | |
| − | :*sequence similarity to your target
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| − | :*size of expected model (length of alignment)
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| − | :*presence or absence of ligands
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| − | :*experimental method and quality of the data set
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| − | Then choose the '''template''' you consider the most suitable and note why you have decided to use this template.
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| − | * Retrieve the most suitable template structure coordinate file from the PDB.
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| − | (0.5 marks)
| + | <br><div style="padding: 5px; background: #EEEEEE;"> |
| | + | *Access the [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=mbp1 information page on Mbp1] at the ''Saccharomyces'' Genome Database and read the summary paragraph on the protein's function! |
| | </div> | | </div> |
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| − | It is not straightforward at all how to number sequence in such a project. The "natural" numbering starts with the start-codon of the full length protein and goes sequentially from there. However, this does not map exactly to other numbering schemes we have encountered. As you know the first residue of the APSES domain as the CDD defines it is not Residue 1 of the Mbp1 protein. The first residue of the e.g. 1MB1 FASTA file '''is''' the first residue of Mbp1 protein, but the last five residues are an artifical His tag. Is H125 of 1MB1 thus equivalent to R125 in MBP1_SACCE? The N-terminus of the Mbp1 crystal structure is disordered. The first residue in the structure is ASN 3, therefore N is the first residue in a FASTA sequence derived from the cordinate section of the PDB file (the <code>ATOM </code> records; whereas the SEQRES records start with MET ... and so on. You need to remember: a sequence number is not absolute, but derived from a particular context.
| + | (If you would like to brush up on the concepts mentioned above, you could study the corresponding chapter in [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.chapter.3432 Lodish's Molecular Cell Biology]. 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.) |
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| − | The homology model will be based on an alignment of target and template. Thus we have to define the target sequence. As discussed in class, PDB files have an explicit and an implied sequence and these do not necessarily have to be the same. To compare the implied and the explicit sequence for the template, you need to extract sequence information from coordinates. One way to do this is via the Web interface for [http://swift.cmbi.ru.nl/servers/html/index.html '''WhatIf'''], a crystallography and molecular modeling package that offers many useful tools for coordinate manipulation tasks.
| + | 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. |
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| − | <div style="padding: 5px; background: #DDDDEE;">
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| − | *Navigate to the '''Administration''' sub-menu of the [http://swift.cmbi.ru.nl/servers/html/index.html WhatIf Web server]. Follow the link to '''Make sequence file from PDB file'''. Enter the PDB-ID of your template into the form field and '''Send''' the request to the server. The server accesses the PDB file and extracts sequence information directly from the <code>ATOM </code> records of the file. The results will be returned in PIR format. Copy the results, edit them to FASTA format and save them in a text-only file. Make sure you create a valid FASTA formatted file! Use this '''implied''' sequence to check if and how it differs from the sequence ...
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| − | :*... listed in the <code>SEQRES</code> records of the coordinate file; | + | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;"> |
| − | :*... given in the FASTA sequence for the template, which is provided by the PDB; | + | ==Retrieve== |
| − | :*... stored in the protein database of the NCBI. | |
| − | : and record your results.
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| − | | |
| − | * In a table, establish how the sequence numbers in the coordinate section of your template(*) correspond to your target sequence numbering.
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| − | | |
| − | (0.5 marks)
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| | </div> | | </div> |
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| − | :(*) <small>These residue numbers are important, since they are referenced e.g. by VMD when you visualize the structure. The easiest way to list them is via the ''Sequence Viewer'' extension of VMD.</small>.
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| − | :<small>Don't do this for every residue individually but define ranges. Look at the correspondence of the first and last residue of target and template sequence and take indels into account. Establishing sequence correspondence precisely is crucially important! For example, when a publication refers to a residue by its sequence number, you have to be able to relate that number to the residue numbers of the model as well as your target sequence.</small>.
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| − | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;">
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| − | ===(1.2) The input alignment (1 mark)=== | + | Much useful information on yeast Mbp1 is compiled at the [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=mbp1 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". |
| − | </div>
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| − | <br>
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| − | The sequence alignment between target and template is the single most important factor that determines the quality of your model. No comparative modeling process will repair an incorrect alignment; it is useful to consider a homology model rather like a three-dimensional map of a sequence alignment rather than a structure in its own right. In a homology modeling project, typically the largest amount of time should be spent on preparing the best possible alignment. Even though automated servers like the SwissModel server will align sequences and select template structures for you, it would be unwise to use these only because they are convenient. You should take advantage of the much more sophisticated alignment methods available. Analysis of wrong models can't be expected to produce right results.
| + | <div style="padding: 5px; background: #EEEEEE; border:solid 1px #AAAAAA;"> |
| − | | + | *Navigate to the NCBI homepage (you probably have bookmarked it anyway) and enter <code>Mbp1 AND "saccharomyces cerevisiae"[organism]</code> as an Entrez query. |
| − | The best possible alignment is usually constructed from a multiple sequence alignment that includes at least '''the target and template sequence''' and other related sequences as well. The additional sequences are an important aid in identifying the correct placement of insertions and deletions. Your alignment should have been carefully reviewed by you and wherever required, manually adjusted to move insertions or deletions between target and template out of the secondary structure elements of the template structure.
| + | *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. |
| − | In the case of Mbp1 genes however, all orthologues we have considered have no indels in the APSES domain regions. Evolutionary pressure on the APSES domains has selected against indels in the more than 600 million years these sequences have evolved independently in their respective species.
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| − | | |
| − | Accordingly, all we need to do is to write the APSES domain sequences one under the other.
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| − | | |
| − | <div style="padding: 5px; background: #DDDDEE;"> | |
| − | * Copy the FASTA formatted sequence for the APSES domain of your organism's Mbp1 orthologue from the sequences [[All_APSES_domains|defined in Assignment 3]] and save it as FASTA formatted text file. This is your '''target''' sequence. Compare this with the FASTA formatted file you have extracted from the PDB coordinate set. This is your '''template''' sequence. Then generate a multi-FASTA formatted file that contains both sequences, and '''pad''' the sequence(s) where required with hyphens as gap characters, so that target and template sequences have exactly the same length and are aligned. Refer to the [[Assignment_4_fallback_data|'''Fallback data''']] if you are not sure about the format. | |
| − | | |
| − | (1 mark)
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| | </div> | | </div> |
| − | <br>
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| − | <br>
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| − | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;">
| + | |
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| − | ==(2) Homology model==
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| − | </div>
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| − | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;">
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| − | === (2.1) SwissModel (1 mark)===
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| − | </div>
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| − | <br>
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| − |
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| − | Access the Swissmodel server at '''http://swissmodel.expasy.org''' . Navigate to the '''Alignment Interface'''.
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| − | <br><div style="padding: 5px; background: #DDDDEE;">
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| − | *Paste your alignment for target and model into the form field. Refer to the [[Assignment_4_fallback_data|'''Fallback Data file''']] if you are not sure about the format. Make sure to select the correct option for the alignment input format on the form.
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| − | :<small>(You have to choose the correct format, and, if e.g. you choose a CLUSTAL format, you have to include a header line and a blank line. In the past we have seen problems with uploading alignments that have not been saved as "text only" and including periods i.e. "." in sequence names of CLUSTAL formatted alignments. Underscores appear to be safe.</small>
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| − |
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| − | * Click '''submit alignment ''' and on the returned page define your '''target''' and '''template''' sequence. For the '''template sequence''' define the PDB ID of the coordinate file. Enter the correct Chain-ID.
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| − | :<small>Recently the PDB has undergone a "remediation" process in which archived coordinate files were altered by the database to conform to new format standards. One of the changes was to assign a chain identifier of "A" to all chains that did not previously have a chain identifier. SwissModel uses a derivative of coordinate sets from the PDB (a dataset they call ExPDB). Apparently the PDB proper and ExPDB have now gone out of synchrony; when I entered the (correct, according to PDB) chain designation "A" for 1MB1, SwissModel rejected the alignment with a nondescript error message. When I entered an underscore "_" instead, which would be the designation for a chain without explicit chain identifier, such as the pre-remidation versio of the coordinates, the alignment was accepted and processed. I have e-mailed SwissModel about the problem; they are in the process of correcting it and may or may not be done while you are working on your assignments. If your template chain has the chain identifier "A" and your alignment gets rejected, try entering entering an underscore instead.</small>
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| − | :<small>'''Enter''' the correct chain ID into the form-field even if you think it already appears there, don't simply accept the preloaded default. There is a bug in SwissModel's parser code that may cause incorrect strings to be sent to the server from that field. I have e-mailed SwissModel about the problem which may or may not be corrected while you are working on your assignments.</small>
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| − | *Click '''submit alignment''' and review the alignment on the returned page. Make sure it has been interpreted correctly by the server. The conserved residues have to be lined up and matching. Then click '''submit alignment''' again, to start the modeling process.
| + | There are several sources for functional domain annotations of proteins. The NCBI has the [http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml Conserved Domain Database], in Europe, the [http://smart.embl-heidelberg.de/ 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. |
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| − | * The resulting page returns information about the resulting model. Save the '''model coordinates''' on your computer. Read the information on what is being returned by the server (click on the red questionmark icon). Paste the Anolea profile into your assignment.
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| − | :<small>Do not paste a screenshot of the result, but copy and paste the image from the Web-page! You do not need to submit the actual coordinate files with your assignment.</small>
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| − | (1 mark) | + | <div style="padding: 5px; background: #EEEEEE;"> |
| | + | *Navigate to the [http://www.uniprot.org/?tab=mapping 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 [http://pir.georgetown.edu/pirwww/search/idmapping.shtml PIR ID-mapping service]. Note the Uniprot accession number you find. (Should this work equally on both sites?) |
| | </div> | | </div> |
| − | <br>
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| − | In case you do not wish to submit the modelling job yourself, or have insurmountable problems when using the SwissModel interface, you may access the result files from the [[Assignment_4_fallback_data|'''Fallback Data file''']]. Document the problems and note this in your assignment.
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| | + | <p> |
| | + | Now navigate to [http://www.uniprot.org '''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. |
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| | + | <p>Note down the SwissProt ID and the UniProtKB Accession Number for yeast Mbp1. |
| | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;"> | | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;"> |
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| − | ==(3) Model analysis== | + | ==Analyse== |
| | </div> | | </div> |
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| | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> | | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> |
| − | === (3.1) The PDB file (1 mark)=== | + | |
| | + | === ''saccharomyces cerevisiae'' Mbp1 - domain annotations=== |
| | </div> | | </div> |
| − | <br>
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| − | Open your '''model''' coordinates in a text-editor (make sure you view the PDB file in a fixed-width font) and consider the following questions: (Alternatively, view the coordinates linked to the [[Assignment_5_fallback_data|'''Fallback Data file''']].)
| + | Now we can analyse Mbp1's domain in SMART, and use this information to annotate the sequence in detail. |
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| − | <br><div style="padding: 5px; background: # DDDDEE;"> | + | <div style="padding: 5px; background: #EEEEEE;"> |
| − | *What is the residue number of the first residue in the '''model'''? What should it be, based on the alignment? If the putative DNA binding region was reported to be residues 50-74 in the Mbp1 protein, which residues of the '''model''' correspond to that? | + | *Navigate to the [http://smart.embl-heidelberg.de/ SMART database], enter the yeast Mbp1 accession number and review the domain features of the protein. |
| − | (1 mark)
| + | *In your assignment, highlight the annotated features in the actual sequence by using the SMART annotations. |
| | </div> | | </div> |
| | | | |
| − | <!-- discuss flagging of loops - setting of B-factor to 99.0 phps. ANOLEA vs. Gromos ... packing vs. energy? -->
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| | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> | | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> |
| − | ===(3.2) First visualization (1 mark)=== | + | |
| | + | === APSES domains === |
| | </div> | | </div> |
| − | <br>
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| − | In assignment 2 you have already studied a Mbp1 structure and compared it with your organism's Mbp1 APSES domain, Since a homology model inherits its structural details from the '''template''', the model should look very similar to the original structure but contain the sequence of the '''target'''.
| + | As you see from the annotations, Mbp1 is a large multidomain protein; it binds DNA through a small domain called the APSES domain and many organisms have more than one transcription factor that has a domain 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 each target organism. |
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| − | <br><div style="padding: 5px; background: #DDDDEE;"> | + | <br> |
| − | *Save your '''model''' coordinates to your harddisk and visualize the structure in VMD. (Alternatively, copy and save the coordinates linked to the [[Assignment_4_fallback_data|'''Fallback Data file''']] to your harddisk.) Make an informative stereo view that shows the general orientation of the helix-turn-helix motif and the "wing", and paste it into your assignment.
| + | Use the NCBI Entrez system to search for the string "apses" in the "Conserved Domains" database and access the entry for the APSES domain. You should find a number of aligned sequences on that page, each with their own GI identifier. |
| − | | |
| − | * Discuss briefly which parts of the model may be unreliable and color these (if any) distinctly in your submitted image.
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| − | (1 mark)
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| | + | <div style="padding: 5px; background: #EEEEEE;"> |
| | + | *Identify the two sequences that come from ''Saccharomyces cerevisiae'' (the Mbp1 and Swi4 APSES domains). |
| | + | *Check whether the NCBI and the SMART definition of the APSES domain in Mbp1 coincide. |
| | + | *Make sure you understand how the sequences displayed on the CDD page and the actual domain sequences differ. <small>Hint: not all sequences are displayed in their full-length.</small> |
| | </div> | | </div> |
| − | <br>
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| − | <br>
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| − | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;">
| + | |
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| − | ==(4) The DNA ligand==
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| − | </div>
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| | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> | | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> |
| | | | |
| − | ===(4.1) Finding a similar protein-DNA complex (1 mark)=== | + | === APSES domain structure === |
| | </div> | | </div> |
| − | <br>
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| − | One of the really interesting questions we can discuss with reference to our model is how sequence variation might be converted into changing DNA recognition sites, and then lead to changed cognate DNA binding sequences. But in order to address this, we would need to add a plausible model for how DNA is bound to APSES domains.
| + | 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! |
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| − | Since there is currently no software available that would accurately model such a complex from first principles, we will base a model of a bound complex on homology modeling as well. This means we need to find a similar structure for which the position of bound DNA is known, then superimpose that structure with our model. This places the DNA molecule into the spatial context of the model we are studying. However, you may remember from the third assignment that the APSES domains in fungi seem to be a relatively small family. And there is no structure available of an APSES domain-DNA complex. How can we find a coordinate set of a strcturally similar protein-DNA complex?
| + | Identify and download the most appropriate coordinate file to study the structure, function and conservation of APSES domains from the PDB. Your choice could be based on: |
| | + | * experimental method (X-ray or NMR) |
| | + | * quality of the structure (resolution, refinement) |
| | + | * size of the structure (number of animo acids for which structure has been determined) |
| | | | |
| − | Remember that homologous sequences can have diverged to the point where their sequence similarity is no longer recognizable, however their structure may be quite well conserved. Thus if we could find similar structures in the PDB, these might provide us with some plausible hypotheses for how DNA is bound by APSES domains. We thus need a tool similar to BLAST, but not for the purpose of sequence alignment, but for structure alignment. A kind of BLAST for structures. Just like with sequence searches, we might not want to search with the entire protein, if we are interested in is a subdomain that binds to DNA. Attempting to match all structural elements in addition to the ones we are actually interested in is likely to make the search less specific - we would find false positives that are similar to some irrelevant part of our structure. However, defining too small of a subdomain would also lead to a loss of specificity: in the extreme it is easy to imagine that the search for e.g. a single helix would retrieve very many hits that would be quite meaningless.
| + | <br><div style="padding: 5px; background: #EEEEEE;"> |
| − | | + | *Record how you have identified the file, what criteria you have used to define whether it is better suited for analysis than others, and paste the <tt>HEADER</tt>, <tt>TITLE</tt>, <tt>COMPND</tt> and <tt>SOURCE</tt> records from the file into your assignment. |
| − | At the '''NCBI''', [http://www.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml VAST] is provided as a search tool for structural similarity search.
| |
| − | | |
| − | At the '''EBI''' there are a number of very well designed structure analysis tools linked off the [http://www.ebi.ac.uk/Tools/structural.html '''Structural Analysis''' page]. As part of its MSD Services, [http://www.ebi.ac.uk/msd-srv/ssm/ '''MSDfold'''] provides a convenient interface for structure searches.
| |
| − | | |
| − | However we have also read previously that the APSES domains are members of a much larger superfamily, the "winged helix" DNA binding domains , of which hundreds of structures have been solved.
| |
| − | | |
| − | <br> | |
| − | | |
| − | [[Image:A5_Mbp1_subdomain.jpg|frame|none|Stereo-view of a subdomain within the 1MB1 structure that includes residues 36 to 76. The color gradient ramps from blue (36) to green (76) and the "wing" is clearly seen as the green pair of beta-strands, extending to the right of the helix-turn-helix motif.]]
| |
| − | | |
| − | <br>
| |
| − | | |
| − | APSES domains represent one branch of the tree of helix-turn-helix (HTH) DNA binding modules. (A recent review on HTH proteins is linked from the resources section at the bottom of this page). Winged Helix domains typically bind their cognate DNA with a "recognition helix" which precedes the beta hairpin and binds into the major groove; additional stabilizing interactions are provided by the edge of a beta-strand binding into the minor groove. This is good news: once we have determined that the APSES domain is actually an example of a larger group of transcription factors, we can compare our model to a structure of a protein-DNA complex. CATH does not provide information on complexes, but we can search the PDB with CATH codes in the following way:
| |
| − | | |
| − | * Access [http://cathwww.biochem.ucl.ac.uk/cgi-bin/cath/GotoCath.pl?cath=1.10.10.10 CATH domain 1.10.10.10]. | |
| − | * Navigate to the [http://www.pdb.org/ PDB home page] and follow the link to [http://www.pdb.org/pdb/search/advSearch.do Advanced Search]
| |
| − | * In the options menu for "Choose a Query Type" select Structure Features → CATH classification. A window will open that allows you to navigate down through the CATH tree. The interface is awkward because it does not display the actual CATH codes along with the class names, but you can view the class names on the CATH page linked above. Click on '''the triangle icons''' before "Mainly Alpha"→"Orthogonal Bundle"→"ARC repressor mutant, subunit A" then click on the link to "winged helix repressor DNA binding domain". As of this writing, this subquery matches 295 structures.
| |
| − | * Click on the (+) button behind the subquery to add an additional query. Select the option "Structure Summary"→"Molecule / Chain type". In the option menus that pop up, select "Contains Protein → Yes", "Contains DNA → Yes""Contains RNA → Ignore". This selects files that contain Protein-DNA complexes.
| |
| − | * Check the box below this subquery to "Remove Similar Sequences at 90% identity" and click on "Evaluate Query". As of this writing, seventy complexes were returned.
| |
| − | * In the left-hand menu, under the Tabulate section, click on the "Collage" function to display icons of the structure files. This is a fast way to obtain an overview of the structures that have been returned. First of all you may notice that in fact not all of the structures are really different, despite selecting only to retrieve dissimilar sequences. This appears to be a deficiency of the algorithm. But you can also easily recognize how the recognition helix inserts into the major groove of most of the structures that were returned (at least those where the domain is not a very small part of a much larger complex). There is one exception: the structure 1DP7 shows how the human RFX1 protein binds DNA in a non-canonical way. We shall use structural superposition of your homology model and two of the winged-helix proteins to decide which mode of DNA binding seems to be more plausible for Mbp1 homologues.
| |
| − | | |
| − | <br><div style="padding: 5px; background: #DDDDEE;">
| |
| − | * Follow the procedure outlined above, from a CATH entry page up to viewing a Collage (or alternatively a tabular view) of the retrieved coordinate files. You can be maximally concise documenting the procedure I have defined above, but do spend a bit of time to understand the key elements of the PDB's advanced search interface.
| |
| − | | |
| − | (1 mark)
| |
| | </div> | | </div> |
| | | | |
| Line 234: |
Line 159: |
| | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> | | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> |
| | | | |
| − | ===(4.2) Preparation and superposition of a canonical complex (1 mark)=== | + | === DNA binding site === |
| | </div> | | </div> |
| − | <br>
| |
| | | | |
| − | The structure we shall use as a reference for the canonical binding mode is the Elk-1 transcription factor. | + | The Mbp1 APSES domain has been shown to bind to DNA and the residues involved in DNA binding have been characterized. ([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]) . In particular the residues between 50-74 have been proposed to comprise the DNA recognition domain. |
| | | | |
| − | [[Image:A5_canonical_wHTH.jpg|frame|none|Stereo-view of the canonical DNA binding mode of the Winged Helix domain family. Shown here is the Elk-1 transcription factor - an ETS DNA binding domain - in complex with a high-affinity binding site (1DUX). Note how the "recognition helix" inserts into the major groove of the DNA molecule. The color gradient ramps from blue (34) to green (84). Note how the first helix of the "helix-turn-helix" architecture serves only to position the recognition helix and makes few interactions by itself.]]
| + | <br><div style="padding: 5px; background: #FFCC99;"> |
| | + | ;Analysis (1 mark) |
| | | | |
| − | The 1DUX coordinate-file contains two protein domains and two B-DNA dimers in one asymmetric unit. For simplicity, let's delete the second copy.
| + | * 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. |
| | | | |
| − | * Access the PDB and navigate to the 1DUX structure explorer page. Download the coordinates to your computer. | + | * 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). |
| − | * Open the coordinate file in a text-editor and delete the coordinates for chains <code>D</code>,<code>E</code> and <code>F</code>; you may also delete all <code>HETATM</code> records and the <code>MASTER</code> record. Save the file with a different name, e.g. 1DUX_monomer.pdb .
| |
| − | * Open VMD and load your homology model. Turn off the axes, display the model as a Tube representation in stereo, and color it by Index. Then load your edited 1DUX file, display this coordinate set in a tube representation as well, and color it by ColorID in some color you like. It is important that you can distinguish easily which structure is which
| |
| − | * You could use the Extensions→Analysis→RMSD calculator interface to superimpose the two strutcures '''IF''' you would know which residues correspond to each other. Sometimes it is useful to do exactly that: define exact correspondences between residue pairs and superimpose according to these selected pairs. For our purpose it is much simpler to use the Multiseq tool (and the structures are simple and small enough that the STAMP algorithm for structural alignment can define corresponding residue pairs automatically). Open the '''multiseq''' extension window, select the check-boxes next to both protein structures, and open the '''Tools→Stamp Structural Alignment''' interface.
| |
| − | * In the "'Stamp Alignment Options'" window, check the radio-button for ''Align the following ...'' '''Marked Structures''' and click on '''OK'''.
| |
| − | * In the '''Graphical Representations''' window, double-click on all "NewCartoon" representations for both molecules, to undisplay them.
| |
| − | * You should now see a superimposed tube model of your homology model and the 1DUX protein-DNA complex. You can explore it, display side-chains etc. and study some of the details of how a transcription factor recognizes and binds to its cognate DNA sequence. However, remember that the model's side-chain orientations have not been experimentally determined but inferred from the template, and that the template's strcture was determined in the absence of bound ligand.
| |
| | | | |
| − | <br><div style="padding: 5px; background: #DDDDEE;">
| + | * 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. |
| − | * Orient and scale your superimposed structures so that their structural similarity is apparent, and the recognition helix can be clearly seen inserting into the DNA major groove. Paste a copy of your image into your assignment. Remark briefly on which parts of the structure appear to superimpose best. Note whether this orientation of a B-DNA double-helix is a plausible model for DNA binding of your Mbp1 orthologue. | |
| | | | |
| − | (1 mark) | + | Paste the images into your assignment in a compressed format. Briefly(!) summarize the VMD forms and parameters you have used. |
| | </div> | | </div> |
| − | <br>
| |
| − |
| |
| | | | |
| | | | |
| − | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;">
| + | DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone. |
| | | | |
| − | ===(4.2) Preparation and superposition of a non-canonical complex (1 mark)===
| + | <br><div style="padding: 5px; background: #FFCC99;"> |
| − | </div>
| + | ;Analysis (2 marks) |
| − | <br> | |
| | | | |
| − | The structure displaying a non-canonical complex between a winged-helix domain and its cognate DNA binding site is the human Regulatory Factor X.
| + | *Report whether this is the case here and which residues might be included. |
| | | | |
| − | [[Image:A5_non-canonical_wHTH.jpg|frame|none|Stereo-view of a non-canonical wHTH-DNA complex, discovered in with the stucture of human Regulatory Factor X (hRFX) binding its cognate X-box DNA sequence (1DP7). Note how the helix that coressponds to the recogition helix in the canonical domain lies across the minor groove whereas the beta-"wing" inserts into the major groove. The color gradient ramps from blue (18) to green (68).]]
| + | *Do the DNA binding residues form a contiguous surface that is compatible with a binding interface? Justify your conclusions. |
| | | | |
| − | The 1DP7 coordinate-file contains only one protein domain and only one B-DNA monomer in its asymmetric unit. This is a file for which we have to generate ''biological unit'' coordinates! Then, for simplicity we will delete the second protein monomer. As you know, there are at least two systems that make the so-called biological units available: the PDB itself, through the Biological Unit file that is accessible via the "Download Files" section on any Structure Explorer page, and the EBI through the PQS service. '''How''' the biological units are stored is subtly different for both cases and for our purpose this small difference is important. The PDB generates additional chins as copies of the original and delineates them with <code>MODEL</code>, <code>ENDMDL</code> records, just like in a multi-structure NMR file. The chain IDs and the atom numbers are the same as the original. The EBI's PQS service creates copies that have distinct atomnumbers and chain IDs. The difference is that the PDB file thus '''contains the same molecule in two different orientations''', wheras the PQS file contains '''two independent molecules'''. This is an important difference when it comes to selecting residues, visualizing and superimposing structures. For VMD, the PQS way of doing things is the right way to go, since by default only the first <code>MODEL</code> will be displayed if several are available.
| + | </div> |
| | | | |
| − | * Access the [http://pqs.ebi.ac.uk/ '''EBI PQS server'''], enter 1DP7 into the '''PDBidcode''' form field and click on '''Submit'''.
| + | |
| − | * On the results page, click on the link under '''1dp7_0''', which is the unique suggestion for a biological unit that the server has identified.
| |
| − | * On the PQS OUTPUT page that is retrieved, click on the '''1dp7.mmol''' link, this will load the PDB formatted coordinate file.
| |
| − | * Save the coordinates as 1DP7_complex.pdb (or some other name that makes sense to you), open it in a text editor, delete the <code>HETATM</code> records from the end and the entire chain "B". Also make sure not to delete any of the <code>TER</code> records for chains "D", "P" or "A". Save the file.
| |
| − | * In the multiseq window, choose File→Import Data, '''Browse...''' to your 1DP7_complex file, select it and click on '''Open'''. Click '''OK''' to load the file.
| |
| − | * Mark all three protein chains by selecting the checkbox next to thier name and again run the STAMP structural alignment.
| |
| − | * In the graphical representations window, double-click again on all cartoon representations that multiseq has generated to undisplay them, undisplay also the Tube representation of 1DUX, create a Tube representatrion for 1DP7, and select a Color by ColorID (a differnet color you like). The resulting scene should look similar to the one you have created above, only with 1DP7 in place of 1DUX and colored differently.
| |
| | | | |
| − | <br><div style="padding: 5px; background: #DDDDEE;"> | + | |
| − | * Orient and scale your superimposed structures so that their structural similarity is apparent, the orientation is similar to the scene generated above and the 1DP7 "wing" can be clearly seen inserting into the DNA major groove. Paste a copy of your image into your assignment. Remark briefly on which parts of the structure appear to superimpose best. Note whether this orientation of a B-DNA double-helix is a plausible model for DNA binding of your Mbp1 orthologue.
| |
| | | | |
| − | (1 mark)
| |
| − | </div>
| |
| − | <br>
| |
| | | | |
| | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> | | <div style="padding: 5px; background: #E9EBF3; border:solid 1px #AAAAAA;"> |
| − | | + | == Onward: the Genome of Interest == |
| − | ===(4.3) Interpretation (2 marks)=== | |
| | </div> | | </div> |
| − | <br>
| |
| − |
| |
| − | In your previous assignment, you have commented on conservation patterns in Mbp1 orthologues. You can refer back to your last results (easier to do), or you can import the APSES domain alignment for Mbp1 proteins and again color by conservation (easier to study) to briefly discuss the following question.
| |
| | | | |
| − | <br><div style="padding: 5px; background: #DDDDEE;">
| + | Up to now, we have looked at the model-organism gene to obtain a baseline of information we are interested in. To move on, we need to access the genome of an organism we are interested in. In this course, the organism of interest is assigned to you. |
| − | * Considering the conservation patterns for Mbp1 orthologues, and assuming that all these orthologues bind DNA in a similar way, which model appears to be more plausible for protein-DNA interactions in APSES domains? Is it the canonical, or the non-canonical binding mode? Discuss briefly what you would expect to find and how this relates to your observations. Distinguish clearly between experimental evidence, computational inference and empirical hypothesis. You are of course welcome to paste detail views (stereo !) of particular sidechains, or surfaces etc. if this helps your arguments. Sometimes a picture is worth many words. But this is not a requirement, we are more interested in evidence-based reasoning than in the form of the presentation.
| |
| | | | |
| − | (2 marks)
| + | The systematic name and strain of a fungus is listed with the [[Group project|project group]] that you have been assigned to. Navigate to the NCBI homepage → "Genomic Biology" → "Fungal Genomes Central" → "Genome Sequencing Projects". This should take you to a tabular view of ongoing and completed fungal genome sequencing projects. Find your organism name in this table. There may be one or more sequencing projects associated with the organism, but there should be only one project for the specific strain. |
| − | </div>
| |
| − | <br> | |
| − | <br> | |
| | | | |
| − | <div style="padding: 5px; background: #BDC3DC; border:solid 1px #AAAAAA;">
| + | Click on the organism name to navigate to the Genome Project information page. |
| | | | |
| − | ==(5) Summary of Resources== | + | <br><div style="padding: 5px; background: #EEEEEE;"> |
| | + | *Review the status of the data you are working with - such as |
| | + | **whether the entire genome is available or only a partial sequence; |
| | + | **How many chromosomes does this genome have? |
| | + | **What is the status of its genome assembly and annotation? |
| | + | **Has the mitochondrial genome been sequenced as well? |
| | + | **Why is this organism deemed important enough to be sequenced? |
| | </div> | | </div> |
| − | <br>
| |
| | | | |
| − | ;Links and background reading | + | |
| | | | |
| − | :* [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/restricted/Peitsch_2002_UseOfModels.pdf '''Review (PDF, restricted)''' Manuel Peitsch on Homology Modeling]
| |
| − | :* [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/restricted/Aravind_2005_HTHdomains.pdf '''Review (PDF, restricted)''' Aravind ''et al.'' Helix-turn-helix domains]
| |
| − | :* [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/restricted/2000_Gajiwala_WingedHelixDomains.pdf '''Review (PDF, restricted)''' Gajiwala & Burley, winged-Helix domains]
| |
| − | :* [[Organism_list_2007|Assigned Organisms]]
| |
| − | :* [http://www.wwpdb.org/documentation/format23/v2.3.html '''PDB file format'''] (see the Coordinate Section if you are unsure about chain identifiers)
| |
| − | :* [http://en.wikipedia.org/wiki/Structural_alignment Wikipedia on '''Structural Superposition'''] <small>(although the article is called "Structural Alignment")</small>
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| | | | |
| − | ;[[Assignment_4_fallback_data|'''Fallback Data page''']] | + | <div style="padding: 5px; background: #D3D8E8; border:solid 1px #AAAAAA;"> |
| − | | + | [End of assignment] |
| − | ;Alignments | + | </div> |
| − | :* [[APSES_domains_MUSCLE|APSES domains MUSCLE aligned]]
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| − | | |
| − |
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| − |
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| | | | |
| − | {{Template:Assignment_Footer}}
| + | If you have any questions at all, don't hesitate to mail me at [mailto:boris.steipe@utoronto.ca boris.steipe@utoronto.ca] or post your question to the [mailto:bch441_2011@googlegroups.com Course Mailing List] |
Note! This assignment is currently active. All significant changes will be announced on the mailing list.
Note! This assignment is currently inactive. Major and minor unannounced changes may be made at any time.
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 9. at 10:00 in the morning.
- Your documentation for the procedures you follow in this assignment will be worth 1 mark.
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. 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, enter the yeast Mbp1 accession number and review the domain features of the protein.
- In your assignment, highlight the annotated features in the actual sequence by using the SMART annotations.
APSES domains
As you see from the annotations, Mbp1 is a large multidomain protein; it binds DNA through a small domain called the APSES domain and many organisms have more than one transcription factor that has a domain 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 each target organism.
Use the NCBI Entrez system to search for the string "apses" in the "Conserved Domains" database and access the entry for the APSES domain. You should find a number of aligned sequences on that page, each with their own GI identifier.
- Identify the two sequences that come from Saccharomyces cerevisiae (the Mbp1 and Swi4 APSES domains).
- Check whether the NCBI and the SMART definition of the APSES domain in Mbp1 coincide.
- 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!
Identify and download the most appropriate coordinate file to study the structure, function and conservation of APSES domains from the PDB. Your choice could be based on:
- experimental method (X-ray or NMR)
- quality of the structure (resolution, refinement)
- size of the structure (number of animo acids for which structure has been determined)
- Record how you have identified the file, what criteria you have used to define whether it is better suited for analysis than others, and paste the HEADER, TITLE, COMPND and SOURCE records from the file into your assignment.
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 (1 mark)
- 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.
Paste the images into your assignment in a compressed format. Briefly(!) summarize the VMD forms and parameters you have used.
DNA binding interfaces are expected to comprise a number of positively charged amino acids, that might form salt-bridges with the phosphate backbone.
- Analysis (2 marks)
- 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.
Onward: the Genome of Interest
Up to now, we have looked at the model-organism gene to obtain a baseline of information we are interested in. To move on, we need to access the genome of an organism we are interested in. In this course, the organism of interest is assigned to you.
The systematic name and strain of a fungus is listed with the project group that you have been assigned to. Navigate to the NCBI homepage → "Genomic Biology" → "Fungal Genomes Central" → "Genome Sequencing Projects". This should take you to a tabular view of ongoing and completed fungal genome sequencing projects. Find your organism name in this table. There may be one or more sequencing projects associated with the organism, but there should be only one project for the specific strain.
Click on the organism name to navigate to the Genome Project information page.
- Review the status of the data you are working with - such as
- whether the entire genome is available or only a partial sequence;
- How many chromosomes does this genome have?
- What is the status of its genome assembly and annotation?
- Has the mitochondrial genome been sequenced as well?
- Why is this organism deemed important enough to be sequenced?
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
