Difference between revisions of "User:Boris/Temp/APB"

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<!-- {{Template:Inactive}} -->
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<div id="APB">
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{{Template:Active}}
 
  
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&nbsp;<br>
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<table width="40%"><tr><td class="l1">&nbsp;</td><td>
  
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__TOC__
+
===Hardware===
 +
<table width="100%">
 +
<tr class="s1"><td class="l1">High performance computing <!-- (... at the bench: GPUs, FPGAs, Clusters) --></td></tr>
 +
<tr class="s2"><td class="l1">Cloud computing</td></tr>
 +
<tr><td class="sp">&nbsp;</td></tr>
 +
</table>
  
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&nbsp;<br>
+
===Systems and Tools===
 +
<table width="100%">
  
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<div style="padding: 5px; background: #A6AFD0;  border:solid 1px #AAAAAA; font-size:200%;font-weight:bold;">
+
<tr class="s1"><td class="l1 mw-collapsible mw-collapsed" data-expandtext="Expand subtopics" data-collapsetext="Collapse">[[Unix]]
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Assignment 3 - Multiple Sequence Alignment
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<div class="mw-collapsible-content">
 +
<table width="100%"><tr class="s2"><td class="l2">[[Unix system administration]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Unix automation]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Program installation]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[wget]]</td></tr></table>
 
</div>
 
</div>
 +
</td></tr>
  
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&nbsp;<br>
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<tr class="s2"><td class="l1">[[Network Configuration]]</td></tr>
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+
<tr class="s1"><td class="l1">[[Apache]]</td></tr>
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{{Template:Preparation|
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<tr class="s2"><td class="l1">[[MySQL]]</td></tr>
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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 people have simply overlooked crucial questions. Sadly, we always get assignments back in which people have not described procedural details. If you did not notice that the above were two different sentences, you are still not reading carefully enough.|
+
<tr class="s1"><td class="l1">[[Tools for the bioinformatics lab]]</td></tr>
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num=3|
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<tr class="s2"><td class="l1">[[GBrowse|GBrowse and LDAS]]</td></tr>
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ord=third|
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<tr><td class="sp">&nbsp;</td></tr>
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due = Monday, October 27. at 10:00 in the morning}}
+
</table>
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;Your documentation for the procedures you follow in this assignment will be worth 1 mark.
 
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&nbsp;<br>
 
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<div style="padding: 2px; background: #F0F1F7;  border:solid 1px #AAAAAA; font-size:125%;color:#444444">
 
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Introduction
 
  
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&nbsp;<br>
+
===Programming===
 +
<table width="100%" >
 +
<tr class="s1"><td class="l1">[[IDE|IDE (Integrated Development Environment)]]</td></tr>
 +
<tr class="s2"><td class="l1">[[Regular Expressions]]</td></tr>
 +
<tr class="s1"><td class="l1">[[Screenscraping]]</td></tr>
  
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;Take care of things, and they will take care of you.
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<tr class="s2"><td class="l1 mw-collapsible mw-collapsed" data-expandtext="Expand subtopics" data-collapsetext="Collapse">[[Perl]]
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:''Shunryu Suzuki''
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<div class="mw-collapsible-content">
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl basic programming]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Perl hash example]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl LWP example]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Perl MySQL introduction]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl OBO parser]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Perl basic programming]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl programming exercises 1]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Perl programming exercises 2]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl programming Data Structures]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Perl references]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl simulation]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Perl: Object oriented programming]]</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[Perl: Ugly programming]]</td></tr></table>
 
</div>
 
</div>
 +
</td></tr>
  
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Much of what we know about a protein's physiological function is based on the '''conservation''' of that function as the organism evolves. We assess conservation by comparison to related proteins. Conservation - or variability - is a consequence of '''selection under constraints''': the multiple effects on an organism's fitness function that are induced through changes to the structural or functional features of a protein. Conservation patterns can thus provide evidence for many different questions: structural conservation among proteins with similar 3D-structures, functional conservation among homologues with comparable roles, peaks of sequence variability that indicate domain boundaries in multi-domain proteins, or amino acid propensities as predictors for protein engineering and design tasks.
+
<tr class="s1"><td class="l1">[[BioPerl]]</td></tr>
 +
<tr class="s2"><td class="l1">[[PHP]]</td></tr>
 +
<tr class="s1"><td class="l1">[[Data modelling]]</td></tr>
 +
<tr class="s2"><td class="l1">BioPython <!-- (scope, highlights, installation, use, support) --></td></tr>
 +
<tr class="s1"><td class="l1">Graphical output <!-- (PNG and SVG) --></td></tr>
 +
<tr class="s2"><td class="l1">[[Autonomous agents]]</td></tr>
 +
</table>
  
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Measuring conservation requires alignment. Therefore a carefully done multiple sequence alignment (MSA) is a cornerstone for the annotation of the essential properties a gene or protein. MSAs are also useful to resolve ambiguities in the precise placement of indels and to ensure that columns in alignments actually contain amino acids that evolve in a similar context. MSAs serve as input for
+
===Algorithms===
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* functional annotation;
+
<table width="100%" >
−
* protein homology modeling;
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<tr class="sh"><td class="l1">Algorithms on Sequences</td></tr>
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* phylogenetic analyses, and
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<tr class="s1"><td class="l2">[[Dynamic Programming]]</td></tr>
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* sensitive homology searches in databases.
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<tr class="s2"><td class="l2">[[Multiple Sequence Alignment]]</td></tr>
 +
<tr class="s1"><td class="l2">[[Genome Assembly]]</td></tr>
  
 +
<tr><td class="sp">&nbsp;</td></tr>
  
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As a first step, we will explore the search and retrieval of fungal proteins that are orthologous to yeast Mbp1, and of the APSES domains they contain. Each student is being assigned one genome-sequenced fungus. Briefly, you will
+
<tr class="sh"><td class="l1">Algorithms on Structures</td></tr>
 +
<tr class="s1"><td class="l2">[[Docking]]</td></tr>
 +
<tr class="s2"><td class="l2">Protein Structure Prediction <!-- ''ab initio'' --></td></tr>
  
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# Collect sequence identifiers for all APSES domain transcription factors in [[Organism list 2011|your assigned organism]];
+
<tr><td class="sp">&nbsp;</td></tr>
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# Retrieve the sequences;
 
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# Perform a multiple sequence alignment with these, and a number of reference domains;
 
−
# Edit the alignment and annotate.
 
  
 +
<tr class="sh"><td class="l1">Algorithms on Trees</td></tr>
 +
<tr class="s1"><td class="l2">Computing with trees <!-- Bayesian approaches for phylogenetic trees, tree comparison) --></td></tr>
  
−
Multiple Sequence Alignment is not a solved, computational problem and a significant number of alignment tools exist, each with different strengths and objectives. It is remarkable that by far the most frequently used MSA algorithm is CLUSTAL, a procedure that was first published for the microprocessors of the late 1980s, surpassed in performance many times, and shown to be significantly inferior to more modern approaches when aligning sequences with 30% identity or less. In this assignment we will encounter various approaches to multiple alignment:
+
<tr><td class="sp">&nbsp;</td></tr>
  
−
* A model-based approach (based on the [[Glossary#PSSM| PSSM]] that PSI-BLAST generates)
+
<tr class="sh"><td class="l1">Algorithms on Networks</td></tr>
−
* Progressive alignments - CLUSTAL and MAFFT
+
<tr class="s1"><td class="l2">Network metrics <!-- (Degree distributions, Centrality metrics, other metrics on topology, small-world- vs. random-geometric controversy) --></td></tr>
−
* Consistency based alignment - T-Coffee and MUSCLE
+
<tr class="s2"><td class="l3">[[Dijkstras Algorithm]]</td></tr>
 +
<tr class="s1"><td class="l3">[[Floyd Warshall Algorithm]]</td></tr>
 +
</table>
  
  
−
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
+
===Communication and collaboration===
−
==(1) Mbp1 homologues==
+
<table width="100%" >
−
</div>
+
<tr class="s1"><td class="l1">[[MediaWiki]]</td></tr>
 +
<tr class="s2"><td class="l1">[[HTML essentials]]</td></tr>
 +
<tr class="s1"><td class="l1">[[HTML 5]]</td></tr>
 +
<tr class="s2"><td class="l1">[[SADI|SADI Semantic Automated Discovery and Integration]]</td></tr>
 +
<tr class="s1"><td class="l1">[[CGI]]</td></tr>
 +
<tr><td class="sp">&nbsp;</td></tr>
 +
</table>
  
 +
===Statistics===
 +
<table width="100%" >
 +
<tr class="s1"><td class="l1">[[Pattern discovery]]</td></tr>
 +
<tr class="s2"><td class="l1">Correlation <!-- (Covariance matrices and their interpretation, application to large problems, collaborative filtering, MIC and MINE) --></td></tr>
 +
<tr class="s1"><td class="l1">Clustering methods <!-- (Algorithms and choice (including: hierarchical, model-based and partition clustering, graphical methods (MCL), flow based methods (RRW) and spectral methods). Implementation in R if possible) --></td></tr>
 +
<tr class="s2"><td class="l1">Cluster metrics <!-- (Cluster quality metrics (Akaike, BIC)–when and how) --></td></tr>
 +
<tr class="s1"><td class="l1">[[Map equation|The Map Equation]] </td></tr>
 +
<tr class="s2"><td class="l1">Machine learning <!-- (Classification problems: Neural Networks, HMMs, SVM..) --></td></tr>
  
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
+
<tr class="s1"><td class="l1 mw-collapsible mw-collapsed" data-expandtext="Expand subtopics" data-collapsetext="Collapse">[[R]]
−
===(1.1) Retrieving sequences===
+
<div class="mw-collapsible-content">
 +
<table width="100%"><tr class="s2"><td class="l2">R plotting</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">[[R programming]]</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">R EDA</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">R regression</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">R PCA</td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">R Clustering</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">R Classification <!-- Phrasing inquiry as a classification problem, dealing with noisy data, machine learning approaches to classification, implementation in R) --></td></tr></table>
 +
<table width="100%"><tr class="s1"><td class="l2">R hypothesis testing</td></tr></table>
 +
<table width="100%"><tr class="s2"><td class="l2">[[Bioconductor]]</td></tr></table>
 
</div>
 
</div>
 +
</td></tr>
  
 +
<tr><td class="sp">&nbsp;</td></tr>
 +
</table>
  
−
In [[Assignment 2]] you retrieved the protein sequences of ''saccharomyces cerevisiae'' [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=6320147 '''Mbp1'''] and defined its APSES (KilA-N) domain. Let us now search for an orthologue of this sequence in ''[[Organism list 2011|Your Organism]]'' More precisely, you should identify prtoteins that fulfill the '''Reciprocal Best Match''' criterion.
+
===Applications===
−
 
+
<table width="100%" >
−
First, we need to '''define the sequence''' you will use to find Mbp1 homologues. Since Mbp1 contains the very widely distributed Ankyrin motifs, a BLAST search with full length sequences will pick up a large number of Ankyrin-repeat containing proteins that are otherwise unrelated to our query. We will instead search for homologues using only the APSES domain as a query. However, the Pfam definition of the APSES domain (or KilA-N family, as it is now called) does not cover the entire length of the domain that has been crystallized. Therefore, we will use the sequence of the crystallized protein instead of the Pfam alignment. One of the results of our analysis will be '''whether APSES domains in fungi all have the same length as the Mbp1 domain, or whether some are indeed much shorter, as sugested by the Pfam alignment.''' To remind you, here is the full sequence of the [http://www.pdb.org/pdb/explore/derivedData.do?structureId=1MB1 1MB1 structure] (Note that the C-terminal His<sub>6</sub> tag that has been added for purification is not part of the Mbp1 protein sequence.) ...
+
<tr class="s1"><td class="l1">[[Data integration]] <!-- Add BioMart: Biodata integration, and data-mining of complex, related, descriptive data --></td></tr>
−
 
+
<tr class="s2"><td class="l1">Text mining <!-- (Use cases, tasks and metrics, taggers, vocabulary mapping, Practicals: R-support, Python/Perl support, others...) --></td></tr>
−
 
+
<tr class="s1"><td class="l1">[[HMMER]]</td></tr>
−
>PDB:1MB1
+
<tr class="s2"><td class="l1">High-throughput sequencing</td></tr>
−
MSNQIYSARYSGVDVYEFIHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRILEKEVLKETHEKVQGGFGKYQGTWVPL
+
<tr class="s1"><td class="l1">Functional annotation <!-- GFF --></td></tr>
−
NIAKQLAEKFSVYDQLKPLFDFTQTDGSASPPPAPKHHHASKVDHHHHHH
+
<tr class="s2"><td class="l1">Microarray analysis <!-- (... in R: differential expression and multiple testing; Loading and normalizing data, calculating differential expression, LOWESS, the question of significance, FWERs: Bonferroni and FDR; SAM and LIMMA) --></td></tr>
−
 
+
<tr><td class="sp">&nbsp;</td></tr>
−
 
 
−
... and, for comparison, this is the corresponding alignment with the Pfam KilA-N model obtained from a '''[http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi RPS-BLAST]''' search of the above sequence against the '''[http://www.ncbi.nlm.nih.gov/cdd/ CDD database]''':
 
−
 
 
−
 
 
−
<span style="color:#700777;">                          10        20        30        40        50        60        70        80</span>
 
−
<span style="color:#700777;">                  ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|</span>
 
−
<b>1MB1</b>          <span style="color:#229922;"> 19 </span><span style="color:#2233cc;">IHSTGS</span><span style="color:#ff4466;">I</span><span style="color:#2233cc;">MK</span><span style="color:#ff4466;">R</span><span style="color:#2233cc;">K</span><span style="color:#ff4466;">KD</span><span style="color:#2233cc;">DWV</span><span style="color:#ff4466;">NAT</span><span style="color:#2233cc;">HIL</span><span style="color:#ff4466;">KAA</span><span style="color:#2233cc;">NFA</span><span style="color:#ff4466;">K</span><span style="color:#888888;">a</span><span style="color:#2233cc;">KRTRI</span><span style="color:#ff4466;">L</span><span style="color:#2233cc;">EK</span><span style="color:#ff4466;">E</span><span style="color:#2233cc;">VL</span><span style="color:#ff4466;">KE</span><span style="color:#2233cc;">TH</span><span style="color:#ff4466;">E</span><span style="color:#2233cc;">KVQ</span><span style="color:#888888;">----------------</span><span style="color:#ff4466;">G</span><span style="color:#2233cc;">GF</span><span style="color:#ff4466;">G</span><span style="color:#2233cc;">KY</span><span style="color:#ff4466;">QGT</span><span style="color:#2233cc;">W</span><span style="color:#ff4466;">V</span><span style="color:#2233cc;">PLNI</span> <span style="color:#229922;">82</span>
 
−
 
−
Cdd:pfam04383  <span style="color:#229922;">  3 </span><span style="color:#2233cc;">YNDFEI</span><span style="color:#ff4466;">I</span><span style="color:#2233cc;">IR</span><span style="color:#ff4466;">R</span><span style="color:#2233cc;">D</span><span style="color:#ff4466;">KD</span><span style="color:#2233cc;">GYI</span><span style="color:#ff4466;">NAT</span><span style="color:#2233cc;">KLC</span><span style="color:#ff4466;">KAA</span><span style="color:#2233cc;">GAT</span><span style="color:#ff4466;">K</span><span style="color:#888888;">-</span><span style="color:#2233cc;">RFRNW</span><span style="color:#ff4466;">L</span><span style="color:#2233cc;">RL</span><span style="color:#ff4466;">E</span><span style="color:#2233cc;">ST</span><span style="color:#ff4466;">KE</span><span style="color:#2233cc;">LI</span><span style="color:#ff4466;">E</span><span style="color:#2233cc;">ELS</span><span style="color:#888888;">kennidvliievenkk</span><span style="color:#ff4466;">G</span><span style="color:#2233cc;">KN</span><span style="color:#ff4466;">G</span><span style="color:#2233cc;">RL</span><span style="color:#ff4466;">QGT</span><span style="color:#2233cc;">Y</span><span style="color:#ff4466;">V</span><span style="color:#2233cc;">HPDL</span> <span style="color:#229922;">81</span>
 
−
 
−
 
−
<span style="color:#700777;">                           90</span>
 
−
<span style="color:#700777;">                  ....*....|....*</span>
 
−
<b>1MB1</b>          <span style="color:#229922;"> 83 </span><span style="color:#ff4466;">A</span><span style="color:#2233cc;">KQL</span><span style="color:#ff4466;">A</span><span style="color:#888888;">----</span><span style="color:#2233cc;">EK</span><span style="color:#ff4466;">F</span><span style="color:#2233cc;">SVY</span> <span style="color:#229922;">93</span>
 
−
 
−
Cdd:pfam04383  <span style="color:#229922;"> 82 </span><span style="color:#ff4466;">A</span><span style="color:#2233cc;">LAI</span><span style="color:#ff4466;">A</span><span style="color:#888888;">swis</span><font  color="#2233cc;">PE</span><span style="color:#ff4466;">F</span><span style="color:#2233cc;">ALK</span> <span style="color:#229922;">96</span>
 
−
 
 
−
 
 
−
As you can see, the Pfam alignment is 18 amino acids shorter at the N-terminus and 31 amino acids shorter at the C-terminus.
 
−
 
 
−
 
 
−
;Find APSES domain proteins in your organism:
 
−
 
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
#Access the [[Organism list|organism list]] and identify the species that has been assigned to you.
 
−
#Navigate to the [http://www.ncbi.nlm.nih.gov '''NCBI's main page'''].
 
−
#Under resources, follow the link to [http://www.ncbi.nlm.nih.gov/guide/genomes-maps/ '''Genomes &amp; Maps'''].
 
−
#In the '''Resources''' section, follow the link to [http://www.ncbi.nlm.nih.gov/sites/genome '''Genomes'''].
 
−
#In the side-bar of that page, follow the link to [http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=euk '''Genomic BLAST (Eukaryotic)'''].
 
−
#Paste the FASTA sequence of the structurally defined APSES domain (e.g. from [http://www.pdb.org/pdb/explore/derivedData.do?structureId=1MB1 1MB1]) into the search field (excluding the His-tag, of course), set the parameters correctly for a '''Protein''' search against '''Protein''' sequences using '''blastp'''. Then find your [[Species list|assigned species]] in the list and check the box next to its name. Run the search. Remember to record the parameters for your search. I expect you to understand what the parameters are that need to be recorded in order to make this search reproducible.
 
−
#On the next screen, check the box next to '''Format for: PSI-BLAST'''. Then click on '''View Report''' to show the results of the first PSI-BLAST iteration.
 
−
#Iterate the PSI-BLAST search until convergence (i.e. until no more '''new''' sequences are added); make sure to include only sequences for which the E-value is small (smaller than about 10e-03 should be safe). If this step does not work for you or the results are not what you expect, please contact your TA right away.
 
−
 
 
−
*Note: Please spend a little time on each page to understand its contents. <small>Ask, if the page contains resources or features you don't understand. Think about what you are doing. If you simply click on the links I provide, you will miss the opportunity to understand how the resources fit into the workflow you are working on, and to be able to execute similar processes yourself. Questions on page contents can potentially appear on quizzes and exam.</small>
 
−
</div>
 
−
 
 
−
 
 
−
Familiarize yourself with the '''output form''' you obtain, this is by far the most frequently used bioinformatics result page. You may want to refer to the [http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/new_view.html NCBI explanation].
 
−
 
 
−
Here is a list of things to look for, all of which I expect you to know and understand. (However you do not need to comment on these points in your submission.)
 
−
 
 
−
;On the alignment image:
 
−
*What do the different colored bars mean?
 
−
*What is the information you get when you "mouse-over" a colored bar on the alignment image.
 
−
*What happens when you click on one of the bars?
 
−
 
 
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;In the description list:
 
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*Where does the link next to an identifier take you?
 
−
*Where does the link in the "score" column take you?
 
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*What does the icon at the end of each row mean? What other icons could appear there? <!-- cf. [http://www.ncbi.nlm.nih.gov/staff/tao/URLAPI/new_view.html] -->
 
−
 
 
−
;In the alignment section:
 
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*What happens if you select three or four matches and then click '''Get selected sequences'''?
 
−
*Why is there sometimes more than one alignment result given with one sequence identifier?
 
−
*What do the alignment metrics mean:
 
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**Score?
 
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**Expect (E-value)?
 
−
**Identities?
 
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**Positives?
 
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**Gaps?
 
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*What is the alignment length?
 
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*Which sequence is labeled '''Query''' and which one is labelled '''Sbjct'''?
 
−
 
 
−
 
 
−
<!---  CONTINUE HERE  --->
 
−
 
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
 
−
 
 
−
==(2) Mbp1 orthologue==
 
−
</div>
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
 
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
===(2.1) Input data for multiple alignments===
 
−
</div>
 
−
 
 
−
 
 
−
You have identified homologues to yeast Mbp1 in your organism, but which one of these (if any) is an '''orthologue'''?
 
−
 
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*Perform a reciprocal BLAST search with your highest scoring hit and note whether the '''reciprocal best match''' criterion has been fulfilled.
 
−
*Repeat this procedure (yeast &rarr; your organism &rarr; yeast) but restict the query sequence to the Mbp1 '''APSES domain''' that you have defined in Assignment 2.
 
−
</div>
 
−
 
 
−
This should retrieve a number of APSES domain proteins in your organism, apparently related to Mbp1.
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
<div style="padding: 5px; background: #FFCC99;">
 
−
;Analysis (1 mark)
 
−
 
 
−
*Based on your results, comment briefly on whether your organism appears to have an orthologue of the entire Mbp1 gene and/or only an APSES domain orthologous to Mbp1.
 
−
 
 
−
</div>
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
Next, compare the (empirical, local) BLAST alignment with a (optimal, global) Needleman-Wunsch sequence alignment. Use the correct algorithm from the set of [http://www.google.ca/search?hl=en&q=emboss+gui EMBOSS tools]:
 
−
 
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*Retrieve the full-length sequence of the orthologue to yeast Mbp1 in your organism, and generate an optimal global alignment between this and ''S. cerevisiae'' Mbp1. <small>You have to figure out where to [http://www.google.ca/search?hl=en&q=emboss+gui find a Web service] that does such alignments, what the name of the algorithm is that you should use and how to define reasonable parameters for the alignment.</small>
 
−
*'''Review''' if and how the alignments are different, or whether the two alignment algorithms have given essentially the same results.
 
−
 
 
−
 
 
−
:<small>'''Note''': When I instruct you to '''review...''', I do not require you to include your conclusions in the submitted assignment. However I expect you to be familiar with the analysis and to be able to answer questions on the process and the conclusions.</small>
 
−
 
 
−
</div>
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
===(2.1) Input data for multiple alignments===
 
−
</div>
 
−
 
 
−
Preparing a set of sequences as input for a multiple sequence alignment usually follows from a BLAST search like the one you have performed above, or perhaps a PSI-BLAST search for added sensitivity. This includes
 
−
 
 
−
*searching a query sequence across a database subset of interest,
 
−
*retrieving orthologue- and or paralogue- sequences,
 
−
*validating BLAST alignments (if needed to distinguish orthologues and paralogues),
 
−
*trimming the sequences to a particular region of interest (if needed to remove non-homologous domains that would otherwise corrupt the alignment), and
 
−
*saving the result as a multi-FASTA formatted file.
 
−
 
 
−
I have generated a reference list of Mbp1 orthologue sequences, using the canonical procedure defined below: (departures from the procedure are noted below the table). Please check whether your orthologue search has identified the same sequence as the one listed in the table. In case the sequence identifiers differ, check whether the actual sequences are the same and in case the actual sequences differ, let me know.
 
−
 
 
−
;Procedure
 
−
 
 
−
# Retrieved the Mbp1 protein sequence by searching [http://www.ncbi.nlm.nih.gov/ Entrez] for <code>Mbp1 AND "saccharomyces cerevisiae"[organism]</code>
 
−
# Clicked on the ''RefSeq tab'' to find the RefSeq ID "<code>[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Protein&list_uids=6320147&dopt=GenPept NP_010227]</code>"
 
−
# Accessed the [http://www.ncbi.nlm.nih.gov/blast '''BLAST'''] form, followed the link to the list of all genomic BLAST databases and clicked on the (B) icon, next to Fungi to navigate to the [http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi Fungi Genomic BLAST page.]
 
−
# Pasted "<code>NP_010227</code>" into the ''query field''. Chose ''Protein'' for both Query and Database, kept default parameters but set the ''Filter'' option to ''none''. Clicked on the check-box of each of the fungal species we have considered in the previous assignment. Run BLAST.
 
−
#On the results page, checked the checkbox next to the alignment to select ''the most significant hit from each organism'' we are studying.
 
−
#Clicked on the "Get selected sequences" button.
 
−
#Separately searched for sequences from organisms that were either not included in the list or for which no hits were reported. Verified all ambiguous cases, as explained in the notes below. 
 
−
#Verified that each of these sequences finds Mbp1 as the best match in the ''saccharomyces cerevisiae'' genome by clicking on each "Blink" ([http://www.ncbi.nlm.nih.gov/sutils/blink.cgi?pid=68465419 <small>click for example</small>]) in the retrieved list. Scrolled down the list to confirm that the '''top hit of a  ''saccharomyces cerevisiae'' protein''' is indeed Mbp1 (<code>NP_010227</code>).
 
−
#Obtained UniProt accessions for all sequences, with a single query using the UniProt [http://www.pir.uniprot.org/search/idmapping.shtml ID mapping service]. This service accepts a comma delimited list of RefSeq IDs, GI numbers or  GenPept accession numbers and returns a list of Uniprot accession numbers.
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
Since it was thus confirmed that each of these sequences is the protein that is most similar to yeast Mbp1 in its respective organism's genome, and that yeast Mbp1 is the most similar yeast protein to each of them, they all fulfil the criterion of a '''reciprocal best match''' with yeast Mbp1. Accordingly we can postulate that this list contains the fungal '''orthologues''' to Mbp1.
 
−
<!-- Clarify: relationship of RBM to orthology -->
 
−
 
 
−
&nbsp;<br>
 
−
 
 
−
<table style="border-left:1px solid #AAAAAA; border-bottom:1px solid #AAAAAA;" cellpadding="10" cellspacing="0">
 
−
 
 
−
<tr style="background: #A6AFD0;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;" colspan="6"><b>Mbp1 and its orthologues</b></td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #BDC3DC;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><b><i>Organism</i></b></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>CODE</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><b>GI</b></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><b>NCBI</b></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><b>Uniprot</b></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><b>Most similar yeast gene</b></td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Aspergillus fumigatus</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>ASPFU</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">70999021</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_754232</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q4WYQ9_ASPFU </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Aspergillus nidulans</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>ASPNI</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">67525393</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_660758</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q5B8H6_EMENI </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Aspergillus terreus</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>ASPTE</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">115391425</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_001213217</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q0CQJ5_ASPTN </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Candida albicans</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>CANAL</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">68465714</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_722925</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q5ANP5_CANAL </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Candida glabrata</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>CANGL</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">50286059</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_445458</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q6FWD6_CANGA </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Coprinopsis cinerea</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>COPCI</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">169861520</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_001837394</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">A8NYC6</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Cryptococcus neoformans</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>CRYNE</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">134110416</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_776035</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q5KHS0_CRYNE </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Debaryomyces hansenii</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>DEBHA</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">50420495</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_458784</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q6BSN6_DEBHA </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Eremothecium gossypii</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>EREGO</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">45199118</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">NP_986147</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q752H3_ASHGO </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Gibberella zeae</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>GIBZE</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">46116756</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_384396</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> UPI000023DBF3 </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Kluyveromyces lactis</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>KLULA</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">50308375</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_454189</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> MBP1_KLULA </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Magnaporthe grisea</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>MAGGR</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">74274844</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">ABA02072 </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q3S405_MAGGR </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1*</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Neurospora crassa</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>NEUCR</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">164424100</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_962967</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q7SBG9 </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Pichia stipitis</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>PICST</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">126275256</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_001386821</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> A3GHD6_PICST </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Saccharomyces cerevisiae</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>SACCE</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">6320147 </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">NP_010227</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> MBP1_YEAST </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Schizosaccharomyces pombe</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>SCHPO</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">19113944</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">NP_593032</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> RES2_SCHPO </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #FFFFFF;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Ustilago maydis</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>USTMA</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">71024227</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_762343</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q4P117_USTMA </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
−
<tr style="background: #E9EBF3;">
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><i>Yarrowia lipolytica</i></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"><code>YARLI</code></td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">50545439</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">XP_500257</td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;"> Q6CGF5_YARLI </td>
 
−
  <td style="border-right:1px solid #AAAAAA; border-top:1px solid #AAAAAA;">Mbp1</td>
 
−
</tr>
 
−
 
 
 
</table>
 
</table>
 +
</td></tr></table>
  
−
<small>Table of yeast Mbp1 orthologues in genome-sequenced fungi. Columns from left to right: Systematic name, organism code (simply a string that lets us identify the organism in alignments), GI number, RefSeq ID (if existing) or GenPept accession, Uniprot accession, most similar yeast protein.
 
−
 
−
 
−
The procedure described above needed to be adapted for some cases and that is to be expected i practice. You should be familiar with exceptions such as the ones described below and know how to deal with them. A good way to do that is to repeat some of the procedures and see if you arrive at the same conclusions.
 
−
 
−
'''Note''': for ''Aspergillus fumigatus'' and ''Aspergillus nidulans'', the top BLAST hit is not the best match. The reason is that the best matching protein has a deletion just C-terminal to the APSES domain. This causes BLAST to split the HSP into two parts, and even though the APSES domain alone has a higher % identity, its E-value turns out to be lower because it is a shorter sequence. Global alignment of each sequence with yeast Mbp1, as well as alignment of only the APSES domains were consistent in showing that for both ''Aspergillus'' species the second highest BLAST score is indeed the most similar protein. The take-home message is that the '''comparison of BLAST scores can be misleading if we apply them to sequences of different length'''. For the record: ''Aspergillus fumigatus'' highest BLAST score is with XP_748947, second highest BLAST score is with XP_754232; the latter has higher global identity (25.7% vs. 22.6%) and higher identity in the APSES domain (55% vs. 45%). ''Aspergillus nidulans'' highest BLAST score is with XP_664319, second highest BLAST score is with XP_660758; the latter has higher global identity (26.7% vs. 22.8%) and higher identity in the APSES domain (59.5% vs. 50.6%). Interestingly, the ''Aspergillus terreus'' orthologue has the same deletion, but it provided the highest BLAST score to begin with.
 
−
 
−
'''Note''': For ''Giberella zeae'' XP_384396  no UniProt ID was returned as cross-reference. EBI-BLAST retrieved  FG04220 which is largely identical, except for short stretches that are absent in GenPept: apparently UniProt has a different gene-model for this protein.
 
−
 
−
'''Note''': The ''Magnaporthe grisea'' protein ABA02072 has greater local C-terminal similarity to the yeast protein Swi6 than to Mbp1, whereas the N-terminal APSES domain is most similar to yeast Mbp1. However a '''global''' Needleman-Wunsch alignment (BLOSUM 30, gaps: 8.0/1.0) shows greater '''overall''' similarity to yeast Mbp1 than to Swi6. Accordingly I consider this an orthologue to Mbp1 even though its database annotation calls ABA02072  the ''M. grisea'' Swi6 homologue.
 
−
 
−
'''Note''': For ''Pichia stipitis'', BLAST finds two very similar sequences in GenPept as candidate Mbp1 orthologues; the RefSeq sequence XP_001386821.1 is translated according to the standard code, the entry EAZ62798.2 is translated according to the alternative nuclear code [http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c#SG12 '''12''']. The question had to be considered which translation appears to be correct. This required looking at the conservation of the residues in question in the BLAST alignment; better conservation indeed supports the alternative code translation.
 
−
 
−
'''Note''': The ''Ustilago maydis'' protein XP_762343 (the protein with the sytematic name UM06196) is only the second-best hit in the original BLAST list as performed on the genomic BLAST page for the organism, however local optimal alignment (EMBOSS water) shows a much higher percentage of identity to yeast Mbp1 in the APSES domain than the top BLAST hit (XP_761485, systematic name UM05338) and global alignment  (after trimming the N- and C- terminal extensions, respectively) also shows a slightly higher degree of similarity for the latter. Accordingly, XP_762343 is considered the Mbp1 orthologue, even though it is the second highest hit according to BLAST. The situation is similar as with the ''Aspergillus'' species, one protein was reported as a single HSP and one protein was broken into two HSPs. This emphasizes the fact that optimal sequence alignments are not entirely equivalent to BLAST alignments. Further, performing the same search against the "'''nr'''" database and applying an '''Organism''' filter for ''Ustilago maydis''  resulted in '''both''' proteins being split and the correct orthologue having the highest BLAST score in the list. This emphasizes the fact that searches in organism databases are not entirely equivalent to searches in the global database, even if the results are filtered.
 
−
 
−
</small>
 
−
 
−
&nbsp;<br>
 
−
 
−
To obtain all FASTA sequences based on a list of identifiers and to save them in a format in which we can use them as input for other programs or services is easy. We can simply paste '''all GI numbers as a comma separated list''' into the Entrez search form and on the results page, select '''Display FASTA''' and '''send to Text'''; then save the contents as a text file. This is a multi-Fasta file, suitable for input into MSA programs.
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*'''Review''' the resulting multi-FASTA file for the  [[All_Mbp1_proteins|'''Mbp1 proteins (linked here)''']] and make sure you understand the procedure that led to it. Depending on your personal learning style you may either carefully review the described procedure, reproduce key steps of the procedure, reproduce the entire procedure paying special attention to the problem cases discussed in the notes, or develop your own procedure. Whatever you do, you must be confident in the end that you could have produced the same input file. (You do not need to submit documentation for this part of the assignment, but you do need to understand the process.)<br>
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
As you have seen from the results of your BLAST searches, Mbp1 orthologues are not the only proteins that contain APSES domains. In order to find all the rest, a PSI-BLAST search was performed using the yeast Mbp1 APSES domain as query. From the list of hits, the APSES domains were extracted and summarized in a file.
 
−
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*'''Review''' the resulting file for the  [[All_APSES_domains|'''APSES domains (linked here)''']] and make sure you understand the procedure that was used in its construction, as above.
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
 
−
==(3) Align and annotate==
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
===(3.1) Review of domain annotations===
 
−
</div>
 
−
 
−
Let us first review some of the features of the yeast Mbp1 protein that we have defined in the second assignment (and some structural features I have compiled from various sources). Below is an annotated yeast Mbp1, compiled according to the following procedure.
 
−
 
−
# Performed [http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi '''CDD'''] search with yeast Mbp1 protein sequence. This retrieves alignments of Mbp1 with the APSES and the ANKYRIN domains. These are profile based alignments and thus they are more reliable than pairwise alignments.
 
−
# Performed  [http://smart.embl-heidelberg.de/ '''SMART'''] search with yeast Mbp1 protein sequence. This retrieved the APSES domain, annotated a number of low-complexity regions and a stretch of coiled coil.
 
−
# Performed a [http://www.ebi.ac.uk/thornton-srv/databases/sas/ '''SAS'''] search with yeast Mbp1 protein sequence. This retrieved pairwise alignments with the structures 1MB1 (APSES) and chain D of 1IKN (ankyrin domains of I<sub>kappa</sub>b), together with their respective secondary structure annotations.
 
−
# Copied GenPept sequence into Word-processor.
 
−
# Transferred annotations of low complexity and coiled-coil regions from SMART.
 
−
# Transferred annotations of APSES secondary structure from SAS (this is a ''direct'' annotation, since the experimentally determined structure 1MB1 is a fagment of of the Mbp1 protein). The central helix that was proposed to be part of the DNA binding region is slightly distorted and SAS annotates a break in the helix, this break was bridged with lowercase "h" in the annotation.
 
−
# Ankyrin domain annotation was not as straightforward. While CDD, SMART and SAS all annotate the same general regions, they disagree in details of the domain boundaries and on the precise alignment. Used the profile-based CDD alignment of 1IKN. Transferred annotations of secondary structure from SAS output for 1IKN to sequence (this is a ''transferred'' annotation, the original annotation was for 1IKN and we assume that it applies to Mbp1 as well).
 
−
 
−
 
−
MBP1_SACCE
 
−
Annotations based on
 
−
- CDD domain analysis,
 
−
- SAS structure annotation and
 
−
- literature data on binding region
 
−
 
−
Keys:
 
−
 
−
C  Coiled coil regions predicted by Coils2 program
 
−
x  Low complexity region
 
−
*  Proposed binding region
 
−
+  positively charged residues, oriented for possible DNA binding interactions
 
−
-  negatively charged residues, oriented for possible DNA binding interactions
 
−
 
−
E  beta strand
 
−
H  alpha helix
 
−
t  beta turn
 
−
 
−
 
−
                  10        20        30        40        50        60
 
−
          MSNQIYSARY SGVDVYEFIH STGSIMKRKK DDWVNATHIL KAANFAKAKR TRILEKEVLK
 
−
1MB1      ----EEEEEt t-EEEEEEEE t-EEEEEEtt ---EEHHHHH HH----HHHH HHHHhhhHHH
 
−
                                                                * *+**-+****
 
−
 
−
                  70        80        90        100        110        120
 
−
          ETHEKVQGGF GKYQGTWVPL NIAKQLAEKF SVYDQLKPLF DFTQTDGSAS PPPAPKHHHA
 
−
1MB1      ---EEE---- tt--EEEE-H HHHHHHHHH- --HHHHtt-        xxx xxxxxxxxxx
 
−
          **+*+***** ****
 
−
 
−
                  130        140        150        160        170        180
 
−
          SKVDRKKAIR SASTSAIMET KRNNKKAEEN QFQSSKILGN PTAAPRKRGR PVGSTRGSRR
 
−
          x                                                                         
 
−
 
−
 
−
                  190        200        210        220        230        240
 
−
          KLGVNLQRSQ SDMGFPRPAI PNSSISTTQL PSIRSTMGPQ SPTLGILEEE RHDSRQQQPQ
 
−
                                                                      xxxxx
 
−
 
−
 
−
                  250        260        270        280        290        300
 
−
          QNNSAQFKEI DLEDGLSSDV EPSQQLQQVF NQNTGFVPQQ QSSLIQTQQT ESMATSVSSS
 
−
          x                                        xx xxxxxxxxxx xxxxxxxxxx
 
−
 
−
 
−
                  310        320        330        340        350        360
 
−
          PSLPTSPGDF ADSNPFEERF PGGGTSPIIS MIPRYPVTSR PQTSDINDKV NKYLSKLVDY
 
−
          xxxxxxx
 
−
 
−
                  370        380        390        400        410        420
 
−
          FISNEMKSNK SLPQVLLHPP PHSAPYIDAP IDPELHTAFH WACSMGNLPI AEALYEAGTS
 
−
ANKYRIN                                -- t----HHHHH HH---HHHHH t-t--t-t--
 
−
 
−
 
−
                  430        440        450        460        470        480
 
−
          IRSTNSQGQT PLMRSSLFHN SYTRRTFPRI FQLLHETVFD IDSQSQTVIH HIVKRKSTTP
 
−
ANKYRIN  t----t---- HHHHHHHH-- -------HHH HHHHHH-ttH HH-----HHH HHHH--tH--
 
−
 
−
 
−
                  490        500        510        520        530        540
 
−
          SAVYYLDVVL SKIKDFSPQY RIELLLNTQD KNGDTALHIA SKNGDVVFFN TLVKMGALTT
 
−
ANKYRIN  HHHHHHHHH- ---------- -----t---- tt---HHHHH HH---HHHHH HHH--t-tt-
 
−
 
−
 
−
                  550        560        570        580        590        600
 
−
          ISNKEGLTAN EIMNQQYEQM MIQNGTNQHV NSSNTDLNIH VNTNNIETKN DVNSMVIMSP
 
−
ANKYRIN  ---t----HH HHHHHH--HH HHH-t--HHH -t----HHHH HHH--tHHHH HHHHHH---t
 
−
 
−
 
−
                  610        620        630        640        650        660
 
−
          VSPSDYITYP SQIATNISRN IPNVVNSMKQ MASIYNDLHE QHDNEIKSLQ KTLKSISKTK
 
−
ANKYRIN  ---tt----H HHHHHH---H HHHHHHH      CCCCCCCC CCCCCCCCCC CCCCC
 
−
 
−
 
−
                  670        680        690        700        710        720
 
−
          IQVSLKTLEV LKESSKDENG EAQTNDDFEI LSRLQEQNTK KLRKRLIRYK RLIKQKLEYR
 
−
                                                    x xxxxxxxxxx xxxxxxx
 
−
 
−
                  730        740        750        760        770        780
 
−
          QTVLLNKLIE DETQATTNNT VEKDNNTLER LELAQELTML QLQRKNKLSS LVKKFEDNAK
 
−
 
−
 
−
                  790        800        810        820        830
 
−
          IHKYRRIIRE GTEMNIEEVD SSLDVILQTL IANNNKNKGA EQIITISNAN SHA
 
−
 
−
 
−
A '''good''' MSA comprises only columns of residues that play similar roles in the proteins' mechanism and/or that evolve in a comparable structural context. Since it is a result of biological selection and conservation, it has relatively few indels and the indels it has are usually not placed into elements of secondary structure or into functional motifs. The contiguous features annotated for Mbp1 are left intact.
 
−
 
−
A '''poor''' MSA has many errors in its columns, they contain residues that actuallly have diffferent functions or structural roles, even though they may look similar to a scoring matrix. It also may have introduced indels in biologically irrelevant positions, to maximize spurious sequence similarities. Some of the features annotated for Mbp1 will be disrupted.
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #EEEEEE;">
 
−
*Produce a similar set of annotations for your Mbp1 orthologue protein.
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
===(3.2) Computing alignments===
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
Multiple sequence alignments are compute-intensive and this used to require downloading and installing software on your own computer. While most tools were available on the Web in principle, many groups have restricted the total number of sequences or the total number of characters to be aligned. The EBI however offers three of the most commonly used tools with few limitations which I have used to produce reference alignments for Mbp1 orthologues and APSES domains.
 
−
 
−
* [http://www.ebi.ac.uk/clustalw/ '''CLUSTAL-W''']  is a progressive alignment program, it is the most popular, most widely referenced MSA algorithm, it is reasonably fast and easy to use. But alignment errors that are made early in the process can't get corrected and thus CLUSTAL is prone to misalign sets of sequences that have poor (<30% ID) local similarity. '''CLUSTAL is no longer considered state-of-the-art''' for carefully done alignments.
 
−
* [http://www.ebi.ac.uk/muscle/ '''MUSCLE'''] essentially starts out from a CLUSTAL like alignment as a draft, then identifies similar groups of sequences from which it calculates profiles, it then re-aligns the group to the profile. This procedure is iterated.
 
−
* [http://www.ebi.ac.uk/t-coffee/ '''T-Coffee'''] is one of my favourites - the tradeoffs appear to be especially well balanced. It too starts from a set of pairwise global alignments, like CLUSTAL, then additionally calculates sets of best local alignments. Global and local alignments are then combined to a similarity matrix and based on this matrix a guide-tree is constructed. This determines the order of steps in which sequences are added to the multiple alignment. A nice feature of T-Coffee is color coded output that allows you to quickly judge the local reliability of the alignment.
 
−
 
−
Multiple sequence alignments were performed for all 18 Mbp1 orthologues. I have posted the reference alignments here. (Of course you are welcome to run an alignment on your own for your own learning experience, or to find an alternative program, but I do not require this for the assignment.)
 
−
 
−
The first alignment was run with CLUSTAL.
 
−
 
−
[[Image:A03_01.jpg|frame|none|Assignment 3, Figure 01<br>
 
−
The guide tree computed by CLUSTAL-W. The algorithm uses this tree to determine the best order for its progressive alignment for the 18 Mbp1 orthologue sequences. This tree is based on a matrix of pairwise distances.]]
 
−
 
−
Subseqently, sequence alignments were performed with T-Coffee and MUSCLE. For these two, the input files were re-ordered to correspond to the order of the CLUSTAL output, and the option to order the alignments according to the ''input sequences'' was chosen on the form. This makes it much easier to compare alignments, since all MSAs are displayed in the same relative order.
 
−
 
−
Finally I have merged the domain annotations for the yeast Mbp1 protein into the output files.
 
−
 
−
The result files are linked here:
 
−
 
−
* [[All_Mbp1_CLUSTAL_annotated|Mbp1 proteins '''CLUSTAL''' aligned]]
 
−
* [[All_Mbp1_MUSCLE_annotated|Mbp1 proteins '''MUSCLE''' aligned]]
 
−
* [[All_Mbp1_T-COFFEE_annotated|Mbp1 proteins '''T-Coffee''' aligned (text version)]] or <small>[http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/resources/T-coffee_scores.html Mbp1 proteins '''T-Coffee aligned'''] (coloured according to scores)</small>
 
−
 
−
Globally speaking, the alignments are quite similar. Let's first look at the common themes, before we discuss details of the results. The  [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/resources/T-coffee_scores.html (score-colored T-COFFEE alignment)] is well suited to look at general relationships between the sequences, since outliers can be easily identified.  For example, if one of the sequences would have a low-scoring domain that aligns poorly to the others of the group, it may be possible that that domain has been acquired in a separate evolutionary event and is not homologous to all others. We would notice an isolated stretch of poorly alignable sequence, i.e. it should be a segment coloured with a low score in a set of otherwise high-scoring segments. Also a gene may have acquired significant lengths of N- or C-terminal extensions which may not be homologous (unless they are the result of an internal duplication).
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*'''Review''' the  [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/resources/T-coffee_scores.html (score-colored T-COFFEE alignment)]. Based on this alignment, how do you feel about our initial assertion that these 18 proteins should be considered orthologous over their entire length? <small>You do not need to discuss this in the assignment but you should study the evidence in the alignment. Note that this question does not ask about the general level of conservation, but about whether significant segments (of about the length of a domain) do not appear related/alignable at all in regions where the rest of the group are reasonably well conserved.</small>
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
 
−
 
−
==(4) Mbp1 orthologues: analysis of full length MSAs==
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
===(4.1)  APSES domains===
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
The APSES domains in all of our Mbp1 orthologues are highly conserved and pretty much any alignment program must be able to align such obviously similar regions.
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*Consider the CLUSTAL, Muscle and T-Coffee alignments of the Mbp1 orthologues.  Orient yourselves as to where the APSES domains are located. For one alignment, refer to the specific residues annoted with (+) or (-) and '''review''' whether the charged residues in the proposed binding region are wholly conserved (marked '''*''') or partially conserved (marked ''':''' or '''.''') across all 18 proteins. Remember that these are surface, solvent exposed residues that would be expected to be highly variable if not constrained for functional reasons. <!-- Sequence variation may indicate variations in binding site -->
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
 
−
===(4.2)  Ankyrin domains===
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
The Ankyrin domains are more highly diverged, the boundaries are less well defined and not even CDD, SMART and SAS agree on the precise annotations. Nevertheless we would hope that a good alignment would recognize homology in that region and that ideally the required ''indels'' would be placed between the secondary structure elements, not in their middle.
 
−
 
−
&nbsp;<br><div style="padding: 5px; background: #EEEEEE;">
 
−
*Compare the distribution of indels in the ankyrin repeat regions of all three alignments. '''Review''' whether the indels in this region are concentrated in segments that connect the helices, or if they are more or less evenly distributed along the entire region of similarity. Think about whether the assertion that ''indels should not be placed in elements of secondary structure'' has merit in each alignment. Recognize that an indel in an element of secondary structure could be interpreted in a number of different ways:
 
−
 
−
**The alignment is correct, the annotation is correct too: the indel is tolerated in that particular case;
 
−
**the alignment algorithm has made an error, the structural anotation is correct: the indel should be moved a few residues;
 
−
**the alignment is correct, the structural annotation is wrong, this is not a secondary structure element after all;
 
−
** both the algorithm and the annotation are probably wrong, but we have no data to improve the situation
 
−
 
−
<small>(NB: remember that the structural annotations have been made for the yeast protein and might have turned out differently for the other proteins...)</small>
 
−
 
−
You should be able to analyse discrepancies in such a structured and systematic way. In particular if you notice indels that have been placed into structurally annotated regions of secondary structure, to consider whether the location of the indel has strong support from aligned sequence motifs, or whether it could apparently be placed  into a different location whithout much loss in alignment quality.
 
−
 
−
Considering the different alignments, please note in your assignment which alignment you consider more reliable regarding the position of indels relative to structural features.
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
 
−
===(4.3)  Other features===
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
'''Aligning''' functional features like ''coiled coil domains'', ''intrinsically disorderd regions'', or ''low complexity regions'' is difficult, since these features are to a large degree a property of the amino acid composition, not of the precise sequence. Thus there may be no recognizable similarity between aligned pairs of amino acids and the correspondence between sequences in such regions may be lost. In such cases, we may be able to detect conserved features in the absence of conserved sequence.
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #FFCC99;">
 
−
;Analysis (1 mark)
 
−
 
−
I have annotated four low complexity regions of the yeast Mbp1 sequence.
 
−
 
−
*Refer to your annotation of your organism's Mbp1 orthologue. Comment on '''one''' of the multiple sequence alignments: does your protein have a similar distribution of low complexity regions as <code>Mbp1_SACCE</code> does, and have these regions been '''aligned''' with the yeast protein by the MSA algorithm? <small>Briefly describe the situation: state whether these segments are found in the same general region, in the same detailed location, or perhaps even conserved in sequence, when you compare them to the ''saccharomyces cerevisiae'' protein.  Backup your conclusions with specific reference to particular elements of the alignment.</small>
 
−
 
−
* Briefly discuss whether this situation implies that disorder in these proteins appears to be a conserved functional feature, i.e. that disorder is selected for in evolution. If this is the case, consider whether the disordered segments appear to be homologous or analogous or whether the data does not allow a conlusion.
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<!-- add at a later time similar analysis of coils via 2ZIP server - conserved feature? [http://2zip.molgen.mpg.de/index.html 2Zip server], also add VMD alignment on ankyrin prototype.
 
−
&nbsp;<br><div style="padding: 5px; background: #EEEEEE;">
 
−
*Task
 
−
</div>
 
−
&nbsp;<br>
 
−
&nbsp;
 
−
-->
 
−
 
−
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
 
−
 
−
==(5) APSES domain homologues: analysis of domain MSAs==
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
You have read how to generate a source sequence file based on the results of a PSI-BLAST search for all APSES domains in fungi. Of course, since PSI-BLAST has detected these sequences due to their high-similarity to a sequence profile, this similarity implies an alignment: a '''model based MSA''' because the sequences are aligned to a model (the sequence profile) and not to each other.
 
−
 
−
To align such highly diverged sequences the MUSCLE server is the tool of choice. For comparison, a CLUSTAL alignment has been computed as well.
 
−
 
−
* The [[APSES_domains_PSI-BLAST| resulting alignment derived from the '''PSI-BLAST''' profile]] as an example of a model-based alignment. <small>Note that PSI-BLAST has not been optimized to work as an alignment program, thus the conclusion that model-based alignments are inferior because this example is a poor alignment is not justified.</small>
 
−
* The [[APSES_domains_CLUSTAL| '''CLUSTAL-W''' alignment]] as an example of a progressive alignment.
 
−
* The [[APSES_domains_MUSCLE| '''MUSCLE''' alignment]] as an example of a consistency-based alignment.
 
−
 
−
If we compare the alignments, we notice immediately that they disagree over siginficant portions of the sequences.
 
−
 
−
&nbsp;<br>
 
−
 
−
<!--
 
−
===(5.0)  Manual improvement===
 
−
 
−
Often errors or inconsistencies are easy to spot, and manually editing an MSA is not generally frowned upon, even though this is not a strictly objective procedure. The main goal of manual editing is to make an alignment biologically more plausible. Most comonly this means to mimize the number of rare evolutionary events that the alignment suggestsand/or to emphasize conservation of known functional motifs. Here are some examples for what one might aim for in manually editing an alignment:
 
−
 
−
* Reduce number of indels
 
−
 
−
From a Probcons alignment:
 
−
0447_DEBHA    ILKTE-K<span style="color:#FF0000;">-</span>T<span style="color:#FF0000;">---</span>K--SVVK      ILKTE----KTK---SVVK
 
−
9978_GIBZE    MLGLN<span style="color:#FF0000;">-</span>PGLKEIT--HSIT      MLGLNPGLKEIT---HSIT
 
−
1513_CANAL    ILKTE-K<span style="color:#FF0000;">-</span>I<span style="color:#FF0000;">---</span>K--NVVK      ILKTE----KIK---NVVK
 
−
6132_SCHPO    ELDDI-I<span style="color:#FF0000;">-</span>ESGDY--ENVD      ELDDI-IESGDY---ENVD
 
−
1244_ASPFU    ----N<span style="color:#FF0000;">-</span>PGLREIC--HSIT  ->  ----NPGLREIC---HSIT
 
−
0925_USTMA    LVKTC<span style="color:#FF0000;">-</span>PALDPHI--TKLK      LVKTCPALDPHI---TKLK
 
−
2599_ASPTE    VLDAN<span style="color:#FF0000;">-</span>PGLREIS--HSIT      VLDANPGLREIS---HSIT
 
−
9773_DEBHA    LLESTPKQYHQHI--KRIR      LLESTPKQYHQHI--KRIR
 
−
0918_CANAL    LLESTPKEYQQYI--KRIR      LLESTPKEYQQYI--KRIR
 
−
 
−
<small>Gaps marked in red were moved. The sequence similarity in the alignment does not change considerably, however the total number of indels in this excerpt is reduced to 13 from the original 22</small>
 
−
 
−
* Move indels to more plausible position
 
−
 
−
From a CLUSTAL alignment:
 
−
4966_CANGL    MKHEKVQ------GGYGRFQ---GTW      MKHEKV<span style="color:#00AA00;">Q</span>------GGYGRFQ---GTW
 
−
1513_CANAL    KIKNVVK------VGSMNLK---GVW      KIKNVV<span style="color:#00AA00;">K</span>------VGSMNLK---GVW
 
−
6132_SCHPO    VDSKHP<span style="color:#FF0000;">-</span>----------<span style="color:#FF0000;">Q</span>ID---GVW  ->  VDSKHP<span style="color:#00AA00;">Q</span>-----------ID---GVW
 
−
1244_ASPFU    EICHSIT------GGALAAQ---GYW      EICHSI<span style="color:#00AA00;">T</span>------GGALAAQ---GYW
 
−
 
−
<small>The two characters marked in red were swapped. This does not change the number of indels but places the "Q" into a a column in which it is more highly conserved (green). Progressive alignments are especially prone to this type of error.</small>
 
−
 
−
* Conserve motifs
 
−
 
−
From a CLUSTAL alignment:
 
−
6166_SCHPO      --DKR<span style="color:#FF0000;">V</span>A---<span style="color:#FF0000;">G</span>LWVPP      --DKR<span style="color:#FF0000;">V</span>A--<span style="color:#FF0000;">G</span>-LWVPP
 
−
XBP1_SACCE      GGYIK<span style="color:#FF0000;">I</span>Q---<span style="color:#FF0000;">G</span>TWLPM      GGYIK<span style="color:#FF0000;">I</span>Q--<span style="color:#FF0000;">G</span>-TWLPM
 
−
6355_ASPTE      --DE<span style="color:#FF0000;">I</span>A<span style="color:#FF0000;">G</span>---NVWISP  ->  ---DE<span style="color:#FF0000;">I</span>A--<span style="color:#FF0000;">G</span>NVWISP
 
−
5262_KLULA      GGYIK<span style="color:#FF0000;">I</span>Q---<span style="color:#FF0000;">G</span>TWLPY      GGYIK<span style="color:#FF0000;">I</span>Q--<span style="color:#FF0000;">G</span>-TWLPY
 
−
 
−
<small>The first of the two residues marked in red is a conserved, solvent exposed hydrophobic residue that may mediate domain interactions. The second residue is the conserved glycine in a beta turn that cannot be mutated without structural disruption. Changing the position of a gap and insertion in one sequence improves the conservation of both motifs.</small>
 
−
 
−
&nbsp;<br>
 
−
 
−
Please consider the following excerpt from the PSI-BLAST alignment:
 
−
 
−
'''Mbp1_SACCE  RILEKEV-LKET-HE--KVQG-GF-GK-----------Y-----------QGTW'''
 
−
MbpA_ASPTE  KTLEKEI-AAGE-HE--KVQG-GY-GK-----------Y-----------QGTW
 
−
MbpC_CANAL  NYFDNEI-LSNLKYF--GSSS-NT-PQ-----------YLDLRKHQNIYLQGIW
 
−
MbpB_CANAL  KLLESTP-KEYQ-QYIKRIRG-GF-LK-----------I-----------QGTW
 
−
MbpA_CANAL  KILEKGV-QQGL-HE--KVQG-GF-GR-----------F-----------QGTW
 
−
Swi4_CANGL  KILEKES-TNMK-HE--KVQG-GY-GR-----------F-----------QGTW
 
−
MbpA_COPCI  KMIDSQPDLAPL-IR--RVRG-GY-LK-----------I-----------QGTW
 
−
MbpA_CRYNE  RVLEREV-QKGE-HE--KVQG-GY-GK-----------Y-----------QGTW
 
−
MbpB_DEBHA  KLLESTP-KQYH-QHIKRIRG-GF-LK-----------I-----------QGTW
 
−
MbpA_DEBHA  KILEKGV-QQGL-HE--KIQG-GY-GR-----------F-----------QGTW
 
−
Swi4_DEBHA  NFLNNEI-LTNT-QY--LSSG-GSNPQFNDLRNHEVRDL-----------RGLW
 
−
Swi4_KLULA  KILEKEA-NEIK-HE--KIQG-GY-GR-----------F-----------QGTW
 
−
Swi4_SACCE  KILEKES-NDMQ-HE--KVQG-GY-GR-----------F-----------QGTW
 
−
Swi4_USTMA  KILEKSI-LTGE-HE--KIQG-GY-GK-----------F-----------QGTW
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
*Find at least one example where this alignment could be manually improved. Show the original version, the improved version, highlight the changes in red and explain your rationale for the change. (1 mark)
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
-->
 
−
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
 
−
===(5.1)  Patterns of residue conservation===
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
Whenever we use a program that optimizes something, we have to be aware of whether the program's objective function actually matches our needs for analysis. For example, if an MSA displays variability in a particular column of residues, this may mean that a residue has changed in evolution - but sometimes the column should have been conserved and the alignment has matched residues with a higher score at the expense of  positions that we believe to be biologically important. MSAs can only take sequence information into account, while we may have complementary information available on structural and functional conservation patterns. This may include secondary structure (gaps should be moved out of regions of secondary structure, where possible), structurally required residues (these are expected to be conserved accross all structurally similar sequences), and functionally conserved residues (these are expected to have a high likelyhood of being conserved within groups of orthologues, but to vary between paralogues).
 
−
 
−
In terms of structural conservation, we expect motif or consistency based alignments to be more accurate since they align to the "big picture". In terms of functional variation we expect progressive alignments to be more accurate, since they align to local similarities.
 
−
 
−
Let us consider the alignments in terms of their biological relevance. I have annotated the ligand-binding residues for the yeast Mbp1 APSES domain in the multiple sequence alignments by color coding the charged residues that putatively could bind DNA <span style="color:#FF0000;">'''red'''</span> (-) and <span style="color:#0066FF;">'''blue'''</span> (+).  Thus these residues label '''columns of the alignment''' in which we expect ''functional'' conservation. I have also highlighted two residues that are associated with important structural features of the APSES domain in <span style="color:#00AA33;">'''green'''</span>. These two residues are <span style="color:#00AA33;">'''G75'''</span>, a glycine required in the third position of a particular type of beta-turn, and <span style="color:#00AA33;">'''W77'''</span>W77, a key component of the domain's hydrophobic core. Thus these two residues label columns in which we expect ''structural'' conservation. Let's assume (''i'') that all the APSES domains fold into similar structures and (''ii'') that they all bind DNA, but (''iii'') they do not necessarily bind the same cognate sequence, as a consequence of the functional diversification of paralogues. This should allow you to discuss the following questions:
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #EEEEEE;">
 
−
Consider any '''one''' of the three APSES domain alignments. 
 
−
*'''Review''' whether the patterns of sequence variation for ''functionally conserved'' residues are compatible with the notion that orthologues have conserved binding specificities and paralogues have acquired new functions by binding to different sequences.
 
−
*'''Review''' whether the patterns of sequence variation for ''structurally conserved'' residues are compatible with the notion that all APSES domains have a common fold.
 
−
 
−
To approach these questions systematically, define (with reference to specific sequences and residues) what you would expect (hypothesis) and whether the alignment supports or contradicts your expectations (observation). We have determined that the sequences labelled as Mbp1 are orthologues, and the other labels were constructed to identify the yeast gene that each sequence is most similar to. This means you may group Mbp1 sequences as orthologues, Swi4, Sok2, and Phd1 sequences are presumably orthologous, and all sequences originating from the same organism are of course groups of paralogues. Labels such as MbpA, MbpB etc. are paralogous to e.g. Mbp1 but not necessarily orthologous to each other.
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
−
===(5.2)  Visualization and analysis of alignment with VMD===
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
VMD offers a very well constructed set of tools for the analyis of sequence and structural conservation: the '''MultiSeq''' extension. In this part of the assignment you will use VMD to analyse and visualize conservation patterns and comment on the alignments the servers have produced. I highly recommend to familiarize yourself with MultiSeq and the developers have produced an [http://www.ks.uiuc.edu/Training/Tutorials/#evolution excellent tutorial on the evolution of tRNA synthetases] to showcase the program's capabilities. However I am not ''requiring'' that you go through the tutorial and we will be using only a subset of the available Multiseq functions. The tool is intuitive enough, beginning to use it should require no more than following the steps below.
 
−
 
−
Proceed through the following steps:
 
−
:(1) Save an alignment of the APSES domains on your computer.
 
−
::(A) Access the MUSCLE alignment of all APSES domains, copy it from the Wiki page and save it on your computer, as a '''text file''' with some convenient filename and the extension .aln . This is a CLUSTAL formatted input file.
 
−
::(B) Edit the file to remove any header lines and lines containing the conservation symbols <code> .:*</code>. Leave the gene-names and aligned sequences as they are. Make sure you are not saving the file in MS-Word binary format (.doc) and that the extension is not changed (depending on how your computer is configured, it may silently append a <code>.txt</code> extension that will cause trouble later on).
 
−
 
−
:(2) Open the Multiseq extension in VMD.
 
−
::(A) start VMD and load the 1MB1 APSES domain structure.
 
−
::(B) choose a stereo representation that will show you the fold of the domain and the sidechains of key residues. For example you could use a Tube representation for the protein backbone and a Licorice representation for the selection <code>((sidechain or type CA) and not element H) and resid 30 to 90</code>.  (And switch the axes display off! The axes carry no information you need).
 
−
::(C) On the VMD Main form navigate to Extensions &rarr; Analysis &rarr; MultiSeq
 
−
::(D) When you run MultiSeq for the first time, you will be asked for a directory in which to store metadata. You can use the default or a directory of your choice; you may subsequently skip all steps that ask you to install "required" databases locally since we will not need them for this task.
 
−
::(E) A window will appear - the ''MultiSeq'' window - it contains the sequence of the APSES domain you are visualizing. MultiSeq will also generate an additional cartoon representation of the structure.
 
−
 
−
:(3) Load the APSES alignment.
 
−
::(A) In the MultiSeq Window, navigate to File &rarr; Import Data...; Choose "From Files" and Browse to the location of the alignment you have saved. The File navigation window gives you options which files to enable: choose to Enable ALN files (these are CLUSTAL formatted multiple sequence alignments).
 
−
::(B) Open the alignment file, click on Ok to Import Data, it will take a short while to load. If the data can't be loaded, the file may have the wrong extension: <code>.aln</code> is required.
 
−
::(C) find the Mbp1_SACCE sequence in the list, click on it and move it to the top of the '''Sequences''' list with your mouse (the list is not static, you can re-order the sequences in any way you like).
 
−
 
−
You will see that the 1MB1 sequence and the APSES domain sequence do not match; at the beginning the structure has extra sequence extending its N-terminus, and in the middle the APSES sequences have gaps inserted.
 
−
 
−
:(4) '''Bring the 1MB1 sequence in register with the APSES alignment'''.
 
−
::(A) MultiSeq supports typical text-editor selection mechanisms. Clicking on a residue selects it, clicking on a row selects the whole sequence. Dragging with the mouse selects several residues, shift-clicking selects ranges, and option-clicking toggles the selection on or off for individual residues. Using the mouse and/or the shift key as required, select the '''entire first column''' of the sequences you have imported.
 
−
::(B) Select Edit &rarr; Enable Editing... &rarr; Gaps only to allow changing indels.
 
−
::(C) Pressing the spacebar once should insert a gap character before the selected column in all sequences. Insert as many gaps as you need to align the beginning of sequences with the corresponding residues of 1MB1 <code>S&nbsp;I&nbsp;M&nbsp;...</code>.
 
−
::(D) Now insert as many gaps as you need into the '''structure''' sequence, to align it completely with the Mbp1_SACCE APSES domain sequence. (Simply select residues in the sequence and use the space bar to insert gaps. <small>(Note: I have noticed a bug that sometimes prevents slider or keyboard input to the MultiSeq window; it fails to ''regain focus'' after operations in a different window. I don't know whether this is a Mac related problem or a more general bug in MultiSeq. When this happens I quit VMD and restore the session from a saved state. It is a bit annoying but not mission-critical.)</small>
 
−
::(E) When you are done, it may be prudent to save the state of your alignment. Use File &rarr; Save Session...
 
−
 
−
:(5) Color by similarity
 
−
::(A) Use the View &rarr; Coloring &rarr; Sequence similarity &rarr; BLOSUM30 option to color the residues in the alignment and structure. This clearly shows you where conserved and variable residues are located and allows to analyze their structural context.
 
−
::(B) You can adjust the color scale in the usual way by navigating to VMD main &rarr; Graphics &rarr; Colors..., choosing the Color Scale tab and adjusting the scale midpoint (0.75 works well for me).
 
−
::(C) Navigate to the Representations window and apply the color scheme to your tube-and-sidechain representation: double-click on the NewCartoon representation to hide it and use '''User''' coloring of your Tube and Licorice representations to apply the sequence similarity color gradient that MultiSeq has calculated. The example below shows in principle what you could expect to see (without sidechains).
 
−
 
−
[[Image:A03_02.jpg|frame|none|Assignment 3, Figure 02<br>
 
−
Stereo view of a tube representation of an APSES domain structure, colored according to residue similarity of all fungal APSES domains as defined in this assignment. A BLOSUM30 similarity matrix was applied and a gradient midpoint of 0.75. The domain is oriented with the putative recognition helix towards the front, left and the "wing" on the right.]]
 
−
 
−
::(D) Now delete all non-Mbp1 sequences from the alignment and recalculate the similarity coloring using only the Mbp1 orthologues. You may want to shift the gradient midpoint to 0.9 or so since overall conservation is much higher. Again study the conservation patterns.
 
−
 
−
[[Image:A03_03.jpg|frame|none|Assignment 3, Figure 03<br>
 
−
Stereo view of a tube representation of an APSES domain structure, colored according to residue similarity of all Mbp1 orthologue APSES domains, as defined in this assignment. A BLOSUM50 similarity matrix was applied and a gradient midpoint of 0.90. The domain is oriented with the putative recognition helix towards the front, left and the "wing" on the right.]]
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #FFCC99;">
 
−
;Analysis (1 mark)
 
−
 
−
*Generate two  parallel stereo views that shows the APSES domain backbone and selected sidechains as described above. One should be colored by sequence similarity among all APSES domains, the other by similarity among only the Mbp1 orthologues. Scale and rotate the structure so that the putative DNA binding domain is easily visible. Paste both views into your assignment in a compressed format, as was explained for Assignment 2.
 
−
 
−
*Briefly discuss what you see (with reference to specific residues and sidechains) and what you conclude about residue conservation in the alignment of all APSES domains. Are the patterns of sequence variation for ''structurally conserved'' residues compatible with the notion that all APSES domains have a common fold?
 
−
 
−
*Briefly discuss how the situation changes when you compare only Mbp1 orthologues with each other. Never mind that overall conservation is higher: does the '''distribution''' of conserved residues in the context of the domain change, and if so, how? Are the patterns of sequence variation for ''functionally conserved'' residues compatible with the notion that all Mbp1 orthologues have a similar function?
 
−
 
−
<small>NB: These are not yes/no questions but require reference to '''specific residues''' and observations.</small>
 
−
 
−
</div>
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
 
−
 
−
==(6) Summary of Resources==
 
−
</div>
 
−
&nbsp;<br>
 
−
 
−
;Links
 
−
:* [http://www.ncbi.nlm.nih.gov/blast '''BLAST''']
 
−
:* [http://www.pir.uniprot.org/?tab=mapping '''Uniprot ID mapping''' service]
 
−
:* [http://www.ncbi.nlm.nih.gov/sutils/blink.cgi?pid=68465419  A '''BLink''' example]
 
−
:* [http://www.ebi.ac.uk/clustalw/ EBI '''CLUSTAL-W''' server]
 
−
:* [http://www.ebi.ac.uk/muscle/ EBI '''MUSCLE''' server]
 
−
:* [http://www.ebi.ac.uk/t-coffee/ EBI '''T-Coffee''' server]
 
−
:* [http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi '''CDD''']
 
−
:* [http://smart.embl-heidelberg.de/ '''SMART''']
 
−
:* [http://www.ebi.ac.uk/thornton-srv/databases/sas/ '''SAS''']
 
−
 
−
;Sequences
 
−
:* [[All_Mbp1_proteins|'''All Mbp1 proteins''']]
 
−
:* [[All_APSES_domains|'''All APSES domains''']]
 
−
 
−
;Alignments
 
−
:'''Mbp1 proteins:'''
 
−
:* [[All_Mbp1_CLUSTAL_annotated|Mbp1 proteins '''CLUSTAL''' aligned]]
 
−
:* [[All_Mbp1_MUSCLE_annotated|Mbp1 proteins '''MUSCLE''' aligned]]
 
−
:* [[All_Mbp1_T-COFFEE_annotated|Mbp1 proteins '''T-Coffee''' aligned (text version)]]
 
−
:* [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/resources/T-coffee_scores.html Mbp1 proteins '''T-Coffee''' aligned (coloured according to scores)]
 
−
 
−
:'''APSES domains:'''
 
−
:* [[APSES_domains_PSI-BLAST|All APSES domains - alignment based on '''PSI-BLAST''' results]]
 
−
:* [[APSES_domains_CLUSTAL|All APSES domains -  '''CLUSTAL-W''' alignment]]
 
−
:* [[APSES_domains_MUSCLE|All APSES domains -  '''MUSCLE''' alignment]]
 
−
 
−
 
−
:'''Further reading'''
 
−
:* [http://bioinformatics.oxfordjournals.org/content/24/3/319.full Moreno-Hagelsieb &amp; Latimer compare Reciprocal Best Match vs. a related concept: Reciprocal Smallest Distance]
 
−
 
−
&nbsp;<br>
 
−
 
−
<div style="padding: 5px; background: #D3D8E8;  border:solid 1px #AAAAAA;">
 
−
[End of assignment]
 
 
</div>
 
</div>
−
 
−
&nbsp;<br>
 
−
 
−
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]
 

Latest revision as of 12:44, 27 September 2015

 

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