Difference between revisions of "BIO Assignment 5 2011"

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Assignment 5 - Phylogenetic Analysis
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Assignment 5 (last: 2011) - Homology modeling
 
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Introduction
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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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&nbsp;
 
&nbsp;
 
&nbsp;
  
;Nothing in Biology makes sense except in the light of evolution.
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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.
:''Theodosius Dobzhansky''
 
</div>
 
  
... but does evolution make sense in the light of biology?
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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.
  
As we have seen in the previous assignments, the Mbp1 transcription factor has homologues in all other fungi, yet - looking at orthologues - this is not always a clear one-to-one mapping of related genes to each other. It appears that various systems of APSES domain transcription factors have evolved independently. Of course this bears directly on our notion of ''function'' - what it means to say that two genes in different organisms have the "same" function. In case two organisms both have an orthologous gene for the same, distinct function, this may be warranted. But what if that gene has duplicated in one of them, and the two paralogues now perform different, related functions in one organism? In order to be able to even ask such questions, we need to understand how we can make the evolutionary history of gene families explicit. This is the domain of '''phylogenetic analysis'''. We can ask questions like: how many paralogues did the cenancestor of a clade possess? Which of these underwent additional duplications in the phylogenesis of the organism I am studying? Did any genes get lost? And - adding additional biological insight to the picture - did the observed duplications lead to the "invention" of new biological systems? When was that? And how did the species benefit from this event?
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''In this assignment you will (1) construct a molecular model of the APSES domain from the Mbp1 orthologue in your assigned species, (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, ''
  
We will develop some of this kind of analysis in this assignment. In the previous assignment you have established which genes are the reciprocally most closely related orthologues to Mbp1 in yeast. In this assignment, we will analyse their evolutionary relationship and compare it to the evolutionary relationship of all fungal APSES domains. The goal is to define families of related transcription factors and their evolutionary history.
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For the following, please remember the following terminology:
  
A number of good tools for phylogenetic analysis exist; ''general purpose packages'' include the (free) PHYLIP package and the (commercial) PAUP package. ''Specialized tools'' for tree-building include Treepuzzle or Mr. Bayes. This assignment is conctructed around programs that are availble in PHYLIP, however you are welcome to use other tools that fulfil a similar purpose if you wish. In this field, researchers consider trees that have been built with ML (maximum likelihood) methods to be more reliable than trees that are built with parsimony methods, or distance methods such as NJ (Neighbor Joining). However ML methods are also much more compute-intensive. Just like with multiple sequence alignments, some algorithms will come closer to guessing the truth and others will not and usually it is hard to tell, which is the more trustworthy of two diverging results. The prudent researcher tries out alternatives and forms her own opinion. Specifically, we may usually assume results that converge, independent of the algorithm, to be more reliable than those that depend strongly on a particular algorithm or details of input data.
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;Target
 
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:The protein that you are planning to model.
But regarding algorithm and rersources: we will take two shortcuts in this assignment (and both shortcuts are things you should not do ''in real life''):
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;Template
 
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:The protein whose structure you are using as a guide to build the model.
One: we will use an '''efficient''' tree-building algorithm, not the best-available one. This is an algorithm which is available on the Web, without the need for you to install software on your own machine. In ''real life'' you would of course use the most accurate algortihm you can lay your hands on, regardless of the resources this requires, since it makes no sense to waste your time on a careful analysis of inaccurate trees. Your supervisor would want it so as well. And if not she, the reviewers of your manuscript.
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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'''.
Two: we will assume the tree the algorithm constructs is ''correct''. In ''real life'' you would establish its reliability with a bootstrap procedure: repeat the tree-building a hundred times with partial data and see which branches and groupings are robust and which depend on the details of the data. But we should still acknowledge that bifurcations that are very close to each other have not been" resolved". Any conscientious reviewer would flag such leniency and send your results back to you for a bootstrapping exercise at the computer. In phylogenetic analysis, not all lines that the program draws are equally trustworthy. Dont take the trees as a given fact just because a program suggests this. Look at the evidence, use your reasoning, and analyse them critically.
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&nbsp;
 
 
In case you want to review concept of trees, clades, LCAs OTUs and the like, I have linked an excellent and very understandable introduction-level article on phylogenetic analysis to the resource section at the bottom of this page.
 
  
&nbsp;
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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.
  
 
{{Template:Preparation|
 
{{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 overlooked marks unnecessarily. If you did not notice that the above did not make sense, you are reading what you expect, not what is written.|
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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.|
 
num=5|
 
num=5|
 
ord=fifth|
 
ord=fifth|
due = Friday, December 7 at 15:00 in the afternoon}}
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due = Monday, December 5. at 12:00 noon}}
  
 
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;Your documentation for the procedures you follow in this assignment will be worth 2 marks - 1 mark for generating the model and 1 mark for the selection/superposition and visualization of protein/DNA complexes.
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<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
==(1) Preparations==
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==(1) Preparation==
 
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===(1.1) Template choice and template sequence===
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The [http://swissmodel.expasy.org/ SWISS-MODEL] server provides several different options for constructing homology models. The easiest is probably the '''Automated Mode''' that requires only a target sequence as input, in this mode the program will automatically choose suitable templates and create an input alignment. I disagree however that that is the best way to use such a service: the reason is that template choice and alignment both may be significantly influenced by biochemical reasoning, and an automated algorithm cannot make the necessary decisions. Should you use a structure of reduced resolution that however has a ligand bound? Should you move an indel from an active site to a loop region even though the sequence similarity score might be less? Questions like that may yield answers that are counter to the best choices an automated algorithm could make. Therefore we will use the '''Alignment Mode''' of Swiss-Model in this assignment, choose our own template and upload our own alignment.
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Template choice is the first step. 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 I have posted 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.
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<div style="padding: 5px; background: #DDDDEE;">
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In assignment 2 you have already searched for structures of APSES domains in the PDB. If you need to repeat this:
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*Use the NCBI BLAST interface to identify all PDB files that are clearly homologous to your target APSES domain.
 +
*In Assignment 2, you have defined the extent of the APSES domain in yeast Mbp1. In Assignment 3, you have aligned reference APSES domains with those you found in your species. In assignment 4 you have confirmed by phylogenetic analysis and ''Recoprocal Best Match'' which of these APSES domain sequences is the closest related orthologue to yeast Mbp1. This sequence is the best candidate for having a conserved function similar to yeast Mbp1. Therefore, this sequence is the '''target''' for the homology modeling procedure.
 +
*Defining a ''template''' means finding a PDB coordinate set that has sufficient sequence similarity to your '''target'' that you can huild a model based on theat '''template'''. To find suitable PDB structures, use your '''target''' sequence as input for a BLAST search, and select Protein Data Bank proteins(pdb) as the '''Database''' you search in. Hits that are homologues are all suitable '''templates''' in principle, but some are more suitable than others. Consider how the coordinate sets differ and which features would make each more or less suitable for creating a homology model; comment briefly on
 +
:*sequence similarity to your target
 +
:*size of expected model (= length of alignment)
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:*presence or absence of ligands
 +
:*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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</div>
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 +
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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 defined by CDD) is not Residue 1 of the Mbp1 protein. The first residue of the 1MB1 FASTA file <small>(one of the related PDB strcuctures)</small> '''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 assigned in a particular context.
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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.
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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&nbsp;&nbsp;</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 ...
 +
 +
:*... listed in the <code>SEQRES</code> records of the coordinate file;
 +
:*... given in the FASTA sequence for the template, which is provided by the PDB;
 +
:*... stored in the protein database of the NCBI.
 +
: and record your results.
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 +
* Establish how the sequence numbers in the coordinate section of your template(*) correspond to your target sequence numbering.
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 +
</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>.
 +
:<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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===(1.1) Preparing Input Files (2 marks)===
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===(1.2) The input alignment===
 
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</div>
 
&nbsp;<br>
 
&nbsp;<br>
  
=====Introduction: Task=====
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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.
For this assignment, we start from the multiple sequence alignments we have constructed in the last assignment. We will edit alignments to make them suitable for phylogenetic analysis. We will construct a tree and we will discuss tree analysis in the end.
 
  
The phylogenetic tree we will construct will contain all APSES domains we have found. In order to '''interpret''' such a tree it is crucial to have some sense of what these domains are, i.e. to cluster them according to their orthologues. Only then can we analyse the tree by asking which subclades mirror the accepted phylogeny of fungi and which ones differ. In the third assignment, you have defined the true orthologues for most of the domains we had previously found with our PSI-BLAST search. (I have filled in the rest.) From this information, I have revised the gene names in the [[APSES_domains_MUSCLE_revised|'''MUSCLE alignment of all APSES domains''']]. When we calculate a phylogenetic tree with these sequences, we should expect orthologues to cluster into the same subclade. Of course, not all fungi have the same number of APSES domain homologues, but from the data we have compiled it should be possible to define their evolutionary history with reference to the other species.
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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.
  
=====Introduction: Principle=====
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In most of the Mbp1 orthologues, we do not observe indels in the APSES domain regions - (and for the ones in which we do see indels, we might suspect that these are actually gene-model errors). 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. To obtain an alignment between the '''template sequence''' and the '''target sequence''' from your species, proceed as follows.  
In order to use molecular sequences for the construction of phylogenetic trees, you have to build a multiple alignment first, then edit it. This is important: all rows of sequences have to contain the exact same number of characters and to hold aligned characters in corresponding positions. Phylogeny programs are not meant to revise an alignment but to analyse evolutionary relationships, given the alignment. Their inferences are made on a column-wise basis and if your columns contain data from unrelated positions, the inferences are going to be questionable.
 
  
The result of the tree construction is a decision about the most likely evolutionary relationships. Fundamentally, tree-construction programs decide which sequences had common ancestors.  
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<div style="padding: 5px; background: #DDDDEE;">
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;In Jalview...
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* Load your Jalview project with aligned APSES domain sequences or recreate it from the Mbp1 orthologue in your species and the APSES domains from the [[Reference APSES domain sequences (reference species)|'''Reference APSES domain page''']] that I prepared for Assignment 4. Include the sequence of your '''template protein''' and re-align.
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* Delete all sequence you no longer need, i.e. keep only the APSES domains of the '''target''' (from your species) and the '''template''' (from the PDB) and choose '''Edit &rarr; Remove empty columns'''. This is your '''input alignment'''.
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* Choose '''File&rarr;Output to textbox&rarr;FASTA''' to obtain the aligned sequences. They should both have exactly the same length, i.e. N- or C- termini have to be padded by hyphens if the original sequences had different length. Save the sequences in a text-file.
  
'''Distance based''' phylogeny programs start by using sequence comparisons to estimate evolutionary distances:
 
* they apply a model of evolution such as a mutation data matrix, to calculate a score for each '''pair''' of sequences,
 
* this score is stored in a "distance matrix" ...
 
* ... and used to estimate a tree that goups sequences with close relationships together. (e.g. by using an NJ, Neigbor Joining, algorithm).
 
They are fast, can work on large numbers of sequences, but are less accurate if genes evolve at different rates.
 
  
'''Parsimony based''' phylogeny programs build a tree that minimizes the number of mutation events that are required to get from a common ancestral sequence to all observed sequences. They take all columns into account, not just a single number per sequence pair, as the Distance methods do. For closely related sequences they work very well, but they construct inaccurate trees when they can't make good estimates for the required number of sequence changes.
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;Using a different MSA program
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* Copy the FASTA formatted sequences of the Mbp1 proteins in the reference  species from the [[Reference APSES domain sequences (reference species)|'''Reference APSES domain page''']].
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* Access e.g. the MSA tools page at the EBI.
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* Paste the Mbp1 sequence set, your '''target''' sequence and the '''template''' sequence into the input form.
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*Run the alignment and save the output.
  
'''ML''', or '''Maximum Lieklihood''' methods attempt to find the tree for which the observed sequences would be the most likely under a particular evolutionary model. They are based on a rigorous statistical framework and yield the most robust results. But they ar also VERY compute intensive and a tree of the size that we are building in this assignment is already almost beyond the resources of common workstations (runs about a day on my computer). However, one may split a large problem into smaller, obvious subtrees (e.g. analysing orthologues as a group, only including a few paralogues for comparison) and then reassemble the smaller trees and in this way even very large problems can become tractable. They also suffer less from "long-branch attraction" - the phenomenon that weakly similar sequences can be grouped inappropriately close together in a tree due to spurious shared diferences distinguishing them from more highly conserved subfamilies.
 
  
Clearly, in order for tree-estimation to work, one must not include fragments of sequence which have evolved under a totally different evolutionary model as all others, such as domain fusion, or large stretches of indels. Thus it is appropriate to edit the sequences and pare them down to a most characteristic subset. The goal is not to be as comprehensive and complete as possible but to input those columns of aligned residues that will best represent the ''true'' phylogenetic relationships between the sequences.
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;By hand
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APSES domains are strongly conserved and have few if any indels. You could also simply align by hand.  
  
=====Introduction: Problems=====
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* Copy the CLUSTAL formatted reference alignment of the Mbp1 proteins in the reference species from the [[Reference APSES domain sequences (reference species)|'''Reference APSES domain page''']].
Gaps are a real problem here, as usual. Strictly speaking, the similarity score of an '''alignment''' program as well as the distance score of a '''phylogeny''' program are not calculated for an ordered ''sequence'', but for a ''sum of independent values'', one for each aligned columns of characters. The order of the columns does not change the score. Hoever in an optimal sequence alignment with gaps, this is no longer strictly true since a one-character gap creation has a different penalty score than a one-character gap extension! Most '''alignment''' programs use a model with a constant gap insertion penalty and a linear gap extension penalty. This is not rigourously justified from biology, but parametrized (or you could say "tweaked") to correspond to our observations. However, most '''phylogeny''' programs, (such as the programs in PHYLIP) do not work in this way. PHYLIP strictly operates on columns of characters and treats a gap character just like a residue with the one letter code "-". Thus gap insertion- and extension- characters get the samescore. For short indels, this '''underestimates''' the distance between pairs of sequences, since any evolutionary model should reflect the fact that gaps are much less likely than point mutations. If the gap is very long though, all events are counted individually as many single substitutions (rather than one lengthy one) and this '''overestimates''' the distance. And it gets worse: long stretches of gaps can make sequences appear similar in a way that is not justified, just because they are identical in the "-" character. It is therefore common to edit gaps in the alignment to one or two character, or to remove them.
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* Open a new file in a text editor.
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* Paste the Mbp1 sequence set, your '''target''' sequence and the '''template''' sequence into the file.
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*Align by hand, replace all spaces with hyphens and save the output.
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</div>
  
=====Introduction: Practice=====
 
In practice, follow the fundamental principle that '''all characters in a column should be related by homology'''. This implies the following rules of thumb:
 
  
:*Remove all stretches of residues in which the ''alignment'' appears ambiguous (not just highly varible, but ambiguous regarding the aligned positions).
+
Whatever method you use: the result should be a multiple sequence alignment in '''multi-FASTA''' format, that was constructed from a number of supporting sequences and that contains your aligned '''target''' and '''template''' sequence. This is your '''input alignment''' for the homology modeling server.
:*Remove all frayed N- and C- termini, especially regions in which not all sequences that are being compared appear homologous and that may stem from unrelated domains.
 
:*Remove all but approximately one column from gapped regions, and all residues N- and C- terminal of the gap in which the alignment appears questionable. ( I would keep one gapped column as a placeholder for a rare and very distinct evolutionary event, rather than simply deleting them all, some researchers remove all gaps).
 
:*Also, consider that neither residues that are completely different between all species, nor residues that are completely conserved are informative for relationship distances.
 
:*If your sequences are too long, you may run out of memory. 60-80 aligned residues should be plenty and if the sequences fit on a single line you will save yourself potential trouble with block-wise vs. interleaved input.
 
  
:<small>(A '''very''' useful trick with Microsoft Word is that you can select blocks of text and entire columns in the document with your mouse: hold the "ALT" key depressed while you click and drag your mouse to select. This will greatly facilitate the preparation of sequences. You can treat that selection as any other selected text, color characters, or delete them. Importantly, you can also cut and paste entire columns! Of course, this will only work as expected if you use a fixed-width font such as Courier. )</small>
 
  
The preparation of the input file of aligned residues, used by the PHYLIP package is straightforward in principle; just carefully follow the instructions in PHYLIP's well written documentation. If you plan to use an outgroup for your tree, it is a good idea to move that to the first line of your alignment, since this is where PHYLIP will look for it by default.
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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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=== (2.1) SwissModel===
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</div>
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&nbsp;<br>
  
Some notes on how to avoid common editing troubles. Copy the sequences frrom the link provided below. Paste them into a document, using the Word "Edit -> Paste special -> Unformatted text". Set the page-setup to "landscape", the font-size to something small, then you can put every sequence into one line. You can replace all paragraph marks ("^p") with (nothing) to remove them, then replace the FASTA header line character ">" with paragraphs ("^p") to separate them by line again. Take special note that your files must not include tab characters. You can use Word to globally replace all tabs (specified as "^t") with a blank, to make sure. Spaces count, so display your alignment in a fixed-width font, such as Courier ("Courier New" on Windows), not a proportional-width font such as Times, Arial, or Helvetica, and ensure all characters in your alignments align as they should. As always, make sure you save your input files as "Text Only".  
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Access the Swissmodel server at '''http://swissmodel.expasy.org''' . Navigate to the '''Alignment Mode''' page.
  
<small>
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&nbsp;<br><div style="padding: 5px; background: #DDDDEE;">
:A note if you are  working on a '''Mac''' and saving input on disk, to run with a locally installe PHYLIP version: here MS Word will play one of its usual [http://en.wikipedia.org/wiki/Shenanigan shenanigans] on you since it writes text files with the old-style OS 9 Carriage Return characters <code>(\r; ASCII 13; hex 0D; CR)</code>. Just by looking at the file, this is quite invisible but such "Carriage returns" are not going to be recognized by PHYLIP and most other self-respecting UNIX based programs. It may not make a difference when you paste your sequences to a Web server; but if you compute things locally it will appear to the program as though everything were in one line). And this can (and did) lead to head-banging rounds of frustration. You need to replace them with '''Linefeed''' resp. '''Newline''' characters <code>(\n; ASCII 10; hex 0A; LF)</code> and you can't even do that within Word(!). Open a UNIX terminal window and navigate to the directory where your files reside. Then type:
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*Paste your alignment for target and model into the form field. Refer to the [[Homology_modeling_fallback_data|'''Fallback Data file''']] if you are not sure about the format. Make sure to select the correct option (FASTA) for the alignment input format on the form.
  
:'''tr "\r" "\n" &lt; infile    &gt; outfile'''
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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 it came from. Enter the correct Chain-ID <small>(usually "A", note: upper-case)</small>.
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:<small>If you run into problems, compare your input to the fallback data. It has worked for me, it will work for you. In particular we have seen problems that arise from "special" characters in the FASTA header like the pipe "<code>|</code>" character that the NCBI uses to separate IDs - keep the header short and remove all non-alphanumeric characters to be safe.</small>
  
:... where outfile is different from infile (careful: if a file by the name of outfile already exists, '''tr''' will cheerfully overwrite it.) Alternatively you could type the following perl one-liner :
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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.
  
:'''perl -e 'while(&lt;&gt;){tr/\r/\n/;print}'  &lt; infile    &gt; outfile'''
+
* 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.
</small>
+
:<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>
 +
</div>
  
  
In your assignment submission, clearly identify the source sequences you are using, as well as the alignment method you have used. Paste your unaltered source alignment into your document, clearly highlight or otherwise color the columns that you have selected, annotate why you have selected them and paste your result as well. Here is an example of what this might look like:
 
  
 +
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
  
[[Image:EditingGuide.jpg|frame|none|(Possible) steps in editing a multiple sequence alignment towards a PHYLIP input file. '''a''': raw alignment (CLUSTAL format); '''b''': sequences assembled into single lines; '''c''': columns to be deleted highlighted in red - 1, 3 and 4: large gaps; 2: uncertain alignment and 5: frayed C-terminus: both would put non-homologous characters into the same column; '''d''': input data for PHYLIP: names for sequences must not be longer than 10 characters, the first line must contain the number of sequences and the sequence length. PHYLIP is very picky about incorrectly formatted input, read the [http://evolution.genetics.washington.edu/phylip/doc/sequence.html PHYLIP sequence format guide].]]
+
==(3) Model analysis==
 +
</div>
 +
&nbsp;
 +
&nbsp;
  
=====Introduction: Web Service and data=====
+
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 +
=== (3.1) The PDB file ===
 +
</div>
 +
&nbsp;<br>
  
You have two choices for completing the assignment: either to use one of the PHYLIP on-line Webservices that generously provide public computing resources, or to download and install the [http://evolution.genetics.washington.edu/phylip.html PHYLIP program package] on your own computer at home. If you choose the former, one of your options is the [http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html '''PHYLIP''' service at the Institut Pasteur] in France.
+
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:
  
<small>I have tried the Pasteur service many times, and it works - however not always entirely without problems. Uninformative errors will occur when your input is too large for the system's memory (like: "sequences not aligned" ... "out of memories" and such) and once, after submitting a number of jobs, the system locked me out, asking me to what an unspecified time until results would be sent by e-mail (which then hasn't happened). Regrettably, this is not documented. However the integration of their services in a logical sequence of steps is good and some of their services are a bit more advanced than plain out of the box PHYLIP. If you rather decide to install PHYLIP, good for you. That is easy to do, well documented, there are much less limitations on memory - but if you don't read and understand the instructions carefully, you may be in for a spell of frustration.</small>
 
  
Either way, I have posted typical input files and result files on the [[Assignment_5_fallback_data|fallback data page]], to allow you to bail out in case technical problems become overwhelming. If you use the data posted here instead of your own, you '''must''' document that fact and explain what you have tried, and why that has failed. The posted data is a fallback, not a shortcut.
+
<br><div style="padding: 5px; background: #DDDDEE;">
 +
*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 your '''model''' correspond to that region?
 +
</div>
 +
<!-- discuss flagging of loops - setting of B-factor to 99.0 phps. ANOLEA vs. Gromos ... packing vs. energy? -->
  
For this assignment, we will use a simple distance based tree construction method. This represents a reasonable compromise between accurracy and speed, especially when applied to moderately dissimilar sequences. In genereal, distance methods include '''two''' steps: (1) calculate a pairwise-distance matrix between sequences, (2) construct a tree, based on the matrix. Thus all the information in the alignment bewtween two pairs of sequences is collapsed into a single number: their pairwise distance. Alternative approaches, parsimony as well as ML based algorithms, take individual columns into account. Parsimony based methods construct inaccurate trees when they can't make good estimates for the required number of sequence changes, if the sequences become too dissimilar. ML based methods are considered the most accurate for dissimilar sequences, however they are also very compute intensive and the full-length APSES domain alignment for 70 sequences run about half a day on my workstation. Thus we will use distance based methods here, specifically the UPGMA variant of the neighbor joining algorithm.
 
  
 +
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 +
===(3.2) First visualization===
 +
</div>
 
&nbsp;<br>
 
&nbsp;<br>
<div style="padding: 5px; background: #DDDDEE;">
 
Prepare an input file that is representative of the APSES domains.
 
  
*Access the [[APSES_domains_MUSCLE_revised|revised MSA for all APSES domains]], linked here (and from the resources section at the bottom of the page). Prepare a PHYLIP formatted input file from this MSA, restricting the number of sequence characters to no more than 70. Read the [http://evolution.genetics.washington.edu/phylip/doc/main.html#inputfiles PHYLIP format documentation] and follow the considerations dicussed above. ([[Assignment_5_fallback_data|See the fallback data in case you get stuck]], but you '''must''' prepare (and document) an input file according to the instructions, even if you end up using the fallback data for whatever reason.) Do not forget to document how you have prepared your input file: define where your source-sequences came from, define which columns you have deleted by highlighing the deleted residues in one sequence, and include your input file in the assignment. (2 marks)
+
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'''.
 +
 
 +
&nbsp;<br><div style="padding: 5px; background: #DDDDEE;">
 +
*Save your '''model''' coordinates to your computer and visualize the structure in VMD. Make an informative (parallel, not cross-eyed!) stereo view that shows the general orientation of the helix-turn-helix motif and the "wing", and paste it into your assignment.
 +
 
 
</div>
 
</div>
 
 
&nbsp;<br>
 
&nbsp;<br>
 
&nbsp;<br>
 
&nbsp;<br>
 +
 +
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
 +
 +
==(4) The DNA ligand==
 +
</div>
 +
&nbsp;
 +
&nbsp;
  
 
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
  
===(1.2) Calculating Trees (3 marks)===
+
===(4.1) Finding a similar protein-DNA complex===
 
</div>
 
</div>
 
&nbsp;<br>
 
&nbsp;<br>
  
 +
One of the really interesting questions we can discuss with reference to our model is how sequence variation might result in changed DNA recognition sites, and then lead to changed cognate DNA binding sequences. In order to address this, we would need to generate a plausible structural model for how DNA is bound to APSES domains.
  
&nbsp;<br>
+
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 structurally similar protein-DNA complex?
<div style="padding: 5px; background: #DDDDEE;">
 
  
*Using the '''protdist''' program of PHYLIP, calculate a distance matrix for the input file you have prepared. ([[Assignment_5_fallback_data|See the fallback data in case you get stuck]]) (1 mark)
+
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.
  
*If you use the PHYLIP Webserver,  select the neighbor joining algorithm from the menu options ('''neighbor''' on the PHYLIP server) and click the button "run the selected program on outfile" ; on the next form, click the button to the "advanced neighbor form", choose the option "UPGMA" and click on the button "run neighbor". When the program is done, select the option '''drawgram''' and click '''Run the selected program on outtree'''. Choose a '''cladogram''' tree-style and a suitable output format (e.g. postscript). Paste the trees 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.  
  
*If you use a locally installed version of PHYLIP use '''neighbor''' with the UPGMA method to construct a tree for both input files. Open the file '''outfile''' in a text-editor, copy and paste the trees into your assignment.
+
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/ '''PDBeFold'''] provides a convenient interface for structure searches.
  
In both cases, the process is: <code>protdist</code> &rarr; <code>neighbor</code> &rarr; <code>drawgram</code>
+
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.
  
:(1 mark for constructing a tree, 1 mark for displaying it).
+
&nbsp;<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.]]
  
</div>
+
&nbsp;<br>
  
&nbsp;<br>
+
APSES domains represent one branch of the tree of helix-turn-helix (HTH) DNA binding modules. (A 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. Superfamilies of such structural domains are compiled in the CATH database. Unfortunately CATH itself does not provide information about whether the structures have been determined as complexes. '''But''' we can search the PDB with CATH codes and restrict the results to complexes. Essentially, this should give us a list of all winged helix domains for which the structure of complexes with DNA have been determined. This works as follows:
&nbsp;<br>
 
  
<div style="padding: 5px; background: #BDC3DCborder:solid 1px #AAAAAA;">
+
* For reference, access [http://cathwww.biochem.ucl.ac.uk/cgi-bin/cath/GotoCath.pl?cath=1.10.10.10 CATH domain 1.10.10.10]; this is the domain you will use to find protein-DNA complexes.
 +
* 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 &rarr; CATH Classification Browser. A window will open that allows you to navigate down through the CATH tree. You can view the Class/Architecture/Topology names on the CATH page linked above. Click on '''the triangle icons''' (not the text) for "Mainly Alpha"&rarr;"Orthogonal Bundle"&rarr;"ARC repressor mutant, subunit A" then click on the link to "winged helix repressor DNA binding domain". Or, just enter "winged helix" into the search field. This subquery should match more than 500 coordinate entries.
 +
* Click on the (+) button behind "Add search criteria" to add an additional query. Select the option "Structure Features"&rarr;"Macromolecule type". In the option menus that pop up, select "Contains Protein &rarr; Yes", "Contains DNA &rarr; Yes""Contains RNA &rarr; Ignore" "Contains DNA/RNA hybrid &rarr; 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 "Submit Query". This query should retrieve more than 90 complexes.
 +
* Scroll down to the beginning of the list of PDB codes and locate the "Generate reports" menu. Under the heading '''Custom reports''' select '''Image collage'''. 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 in most of the the structures the '''recognition helix inserts into the major groove of B-DNA''' (eg. 1BC8, 1CF7). There is one exception: the structure 1DP7 shows how the human RFX1 protein binds DNA in a non-canonical way, through the beta-strands of the "wing". This is interesting since it suggests there is more than one way for winged helix domains to bind to DNA. We can therefore use structural superposition of '''your homology model''' and '''two of the winged-helix proteins''' to decide whether the canonical or the non-canonical mode of DNA binding seems to be more plausible for Mbp1 orthologues.
  
==(2) Analysis==
+
&nbsp;<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 in your documentation for the procedure I have defined above, but I expect you to have spent enough time on this process to understand the key elements of the PDB's advanced search interface.
 
</div>
 
</div>
  
I have constructed a cladogram for the species we are analysing, based on data published for 1551 fungal ribosomal sequences. Such reference tres from rRNA data are a standard method of phylogenetic analysis, supported by the assumption that rRNA sequences are monophyletic and have evolved under comparable selective pressure in all species.
 
  
[[Image:FungiCladogram.jpg|frame|none|Cladogram of fungi studied in the assignments. This cladogram is based on small subunit ribosomal rRNA sequences, and largely follows ''Tehler et al.'' (2003) ''Mycol Res.'' '''107''':901-916. Even though many details of fungal phylogeny remain unresolved, the branches shown here individually appear to have strong support. In a cladogram such as this, the branch lengths are not drawn to any scale of similarity. I have labeled all speciation events so you can refer to these labels in your assignment.]]
+
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
  
In order to study the evolutionary history of the entire gene family you can use the tree you have computed or access the [[APSES_domains_reference_tree|'''APSES domains reference tree''']] here.
+
===(4.2) Preparation and superposition of a canonical complex===
 +
</div>
 +
&nbsp;<br>
  
This is a complicated tree, and it can look impenetrably confusing at first. Here are two principles that will help you make sense of the tree.
+
The structure we shall use as a reference for the '''canonical binding mode''' is the Elk-1 transcription factor.
  
A: '''A gene that is present in an ancestral species, is inherited in all descendent species.''' The gene has to be observed in all OTUs, unless its has been lost (which is a rare event). This means, if a gene is present in two widely divergent species, but in none other of the descendants of the LCA, it is possible that there is some problem with the tree (long branch attraction maybe), or the sequence has been acquired through horizontal gene transfer.
+
[[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.]]
  
B: '''Paralogous genes in an ancestral species should give rise to monophyletic subtrees for each of the genes, in all descendants'''; this means: if the LCA of a branch has e.g. three genes, we would expect three copies of the species cladogram below this branchpoint, one for each of these genes. Each of these subtrees should recapitulate the reference phylogenetic tree of the OTUs, up to the branchpoint of their LCA.
+
The 1DUX coordinate-file contains two protein domains and two B-DNA dimers in one asymmetric unit. For simplicity, you should delete the second copy of the complex from the PDB file. (Remember that PDB files are simply text files that can be edited.)
  
With these two simple principles (you should draw them out on a piece of paper if they do not seem obvious to you), you can probably pry the [[APSES_domains_reference_tree|reference tree of all APSES domains]] apart quite nicely. A few colored pencils and a printout of the tree will help.
+
* Access the PDB and navigate to the 1DUX structure explorer page. Download the coordinates to your computer.
 +
* 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&rarr;Analysis&rarr;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&rarr;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 your '''model''''s side-chain orientations have not been determined experimentally but inferred from the '''template''', and that the template's structure was determined in the absence of bound DNA ligand.
  
 +
&nbsp;<br><div style="padding: 5px; background: #DDDDEE;">
 +
* 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.
  
&nbsp;
+
</div>
 +
&nbsp;<br>
 
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&nbsp;
  
Line 185: Line 273:
 
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<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
  
===(2.1) The Cenancestor's APSES Domains (2 marks)===
+
===(4.2) Preparation and superposition of a non-canonical complex===
 
</div>
 
</div>
&nbsp;<br>
 
  
  
&nbsp;<br><div style="padding: 5px; background: #EEEEEE;">
+
The structure displaying a non-canonical complex between a winged-helix domain and its cognate DNA binding site is the human Regulatory Factor X.
*Identify at least two (more or less) monophyletic subtrees of the tree that recapitulate (more or less) the species tree. Write down their branch-point numbers. In one of these subtrees, define the correspondence between the letters in the species tree and the branchpoints in the gene tree '''as far as possible'''. (The correspondence is not going to be exact. ) (1 mark)
+
 
 +
[[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 coresponds to the recognition 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).]]
 +
 
 +
 
 +
Before we can work with this however, we have to fix an annoying problem caused by the way the PDB stores replicates in biological assemblies. The PDB generates additional chains as copies of the original and delineates them with <code>MODEL</code> and <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 PDB file thus contains the '''same molecule in two different orientations''', not '''two independent molecules'''. This is an important difference regarding how such molecules are displayed by VMD. If you were to use the biological unit file of the PDB, VMD does not recognize that there is a second molecule present and displays only one. We have to edit the file to merge the two molecules by removing the MODEL and ENDMDL records - and while we're editing the file we'll also remove unneeded heteroatoms and the second copy of the protein chain (which we don't need, we need only the second B-DNA strand). But then we end up with residues that have '''exactly the same residue number''' in the same file. That won't work for visualization, since the program expects residue numbers to be unique, therefore we have to renumber the residues. Here's how:
  
<!-- Discuss briefly how many APSES domain proteins the fungal cenancestor appears to have posessed and by which sequence of gene loss or gene duplications the APSES domains in "your" organism appear to have arisen. (This is a straightforward synthesis based on what you have done above, by referring to labelled nodes in the reference cladogram.) -->
+
* On the structure explorer page for 1DP7, select the option '''Download Files''' &rarr; '''Biological Assembly'''.
 +
* Dowload, save and uncompress the file.
 +
* Open the file in a text editor.
 +
* Delete both <code>MODEL</code> and both <code>ENDMDL</code> records.
 +
* Also delete all <code>HETATM</code> records for <code>HOH</code>, <code>PEG</code> and <code>EDO</code>, as well as the entire second protein chain and the <code>MASTER</code> record. The resulting file should only contain the DNA chain and its copy and one protein chain. Save the file with a new name.
 +
* Access the [http://swift.cmbi.ru.nl/servers/html/index.html '''Whatif Web interface'''] and click on '''Administration''' and '''Renumber a PDB File from 1'''. Upload your edited file, access the results and save the file.
 +
* Open the renumbered file with VMD. You should see '''one protein chain''' and a '''B-DNA double helix'''. Switch to stereo viewing and spend some time to see how '''amazingly beautiful''' the complementarity between the protein and the DNA helix is (you might want to display ''chain P'' and ''chain D'' in separate representations and color the DNA chain by ''Position'' &rarr; ''Radial'' for clarity) ... in particular, appreciate how Arginine 76 interacts with the base of Guanine 92!
 +
* Then clear all molecules
 +
* In VMD, open '''Extensions&rarr;Analysis&rarr;MultiSeq'''. 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.
 +
* 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. Choose '''File&rarr;Import Data''', browse to your directory and load:
 +
** Your model;
 +
** The 1DUX complex;
 +
** The 1DP7 complex.
 +
* Mark all three protein chains by selecting the checkbox next to their name and run the STAMP structural alignment.
 +
* In the graphical representations window, double-click on the cartoon representations that multiseq has generated to undisplay them, also undisplay the Tube representation of 1DUX. Then create a Tube representation for 1DP7, and select a Color by ColorID (a different color that 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.
  
* Discuss briefly if there are features of the gene tree that are systematically inconsistent with the reference cladogram: choose '''one''' species from the gene tree that violates the expectation from the species tree and check what it's close neighbors are for the other parts of the tree. Do these sequences always appear more closely or more distantly related than they should be? (1 mark)
+
&nbsp;<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.
 
</div>
 
</div>
&nbsp;<br>
+
 
&nbsp;
 
  
 
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
 
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
  
===(2.3) Unraveling the Mbp1 clade (2 marks)===
+
===(4.3) Coloring by conservation===
 
</div>
 
</div>
&nbsp;<br>
 
  
If we consider the Mbp1 clade (the clade containing all 16 of our Mbp1 orthologues), descending from branchpoint '''115''', we note that it contains 16 additional genes, one from each of the species we have studied, however both subtrees are polyphyletic within the clade. (In what follows I will refer to subgroups of the tree as ''Clades'' and label them with their branch number in this reference tree.) It is thus tempting to speculate that this subtree, ''Clade 115'' is actually a mix-up of two ancestral genes, one of them an orthologue to Mbp1, the other to a different gene - perhaps Swi4. If this were true, there would be three possible explanations for the polyphyletic tree: (i) either we have mislabeled the orthologues, or (ii) we have constructed an incorrect tree, or (iii) our model of how these genes have evolved is wrong. Actually, it is rather obvious when we compare the clades that branch off at 117 and 128, why this is not a simple case of mislabeling the genes ...
+
With the superimposed coordinates, you can begin to get a sense whether either or both binding modes could be appropriate for a protein-DNA complex in your Mbp1 orthologue. But these are geometrical criteria only, and the protein in your species may be flexible enough to adopt a different conformation in a complex, and different again from your model. A more powerful way to analyze such hypothetical complexes is to look at conservation patterns. With VMD, you can import a sequence alignment into the MultiSeq extension and color residies by conservation. The protocol below assumes
 +
 
 +
*You have prealigned the reference Mbp1 proteins with your species' Mbp1 orthologue;
 +
*You have saved the alignment in a CLUSTAL format.
 +
 
 +
You can use Jalview or any other MSA server to do so. You can even do this by hand - there should be few if any indels and the correct alignment is easy to see.
 +
 
 +
 
 +
;Load the Mbp1 APSES alignment into MultiSeq.
 +
 
 +
:(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 <code>ALN</code> files (these are CLUSTAL formatted multiple sequence alignments).
 +
:(B) Open the alignment file, click on '''Ok''' to import the data, it will take a short while to load. If the data can't be loaded, the file may have the wrong extension: .aln 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 N-terminus the sequence that corresponds to the PDB structure has extra residues, and in the middle the APSES sequences may have gaps inserted.
 +
 
 +
;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 I M ...</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. (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.)
 +
:(E) When you are done, it may be prudent to save the state of your alignment. Use '''File &rarr; Save Session...'''
 +
 
 +
;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.
 +
:(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.  
  
&nbsp;<br><div style="padding: 5px; background: #EEEEEE;">
+
&nbsp;<br><div style="padding: 5px; background: #DDDDEE;">
*Explain briefly why it is unlikely that the clades at branchpoints 117 and 128 are each descendants of a single cenancestral gene. (1 mark)
+
* Once you have colored the residues of your model by conservation, create another informative stereo-image and paste it into your assignment.
 
</div>
 
</div>
&nbsp;<br>
 
  
Could this point to a problem with the columns we have selected for the tree building? Have we biased our input data and then constructed an incorrect tree? To test this, a revised tree was constructed, taking the genes from ''Clade 115'', realigning them and constructing a new tree. You can have a look at the [[Revised_Mbp1_APSES_domain_tree| '''revised Mbp1 APSES domain tree''']], and the procedure - or you can just take my word for it, the trees are substantially similar. Apparently the precise choice of columns has a minor effect on the results, as it should have. We should note in general however that '''branch points that are close together in time may be poorly resolvable, while branch points that are separated by longer evolutionary distances are more reliable'''. In summary, save for some inaccuracies, we have no reason to believe the tree is fundamentally incorrect.
 
  
So what about or model of evolution? What do we actually expect?
 
  
As we had written above, we expect to see anumber of more or less faithful copies of the species tree, for each of gene in the cenancestor (or LCA for later duplications).
+
<div style="padding: 5px; background: #E9EBF3;  border:solid 1px #AAAAAA;">
  
And as you see, this is '''not''' exactly what we find in ''Clade 115''. What we '''do''' find however is that ''Clade 115'' reflects our expectations in part. ''Clade 123'' pretty well recapitulates the ''Saccharomycotina'', as does ''Clade 118'', and when we consider ''Clade 128'', apart from some "noise" we see both the ''Dikaryomycota'' (in ''Clade 142'') reasonably grouped together, as well as the ''Euascomycotina'' (in ''Clade 129") - in fact the most striking departure from our expected tree for Mbp1 is the placement of ''Clade 123'', which we would expect attached below branch 128, not above it where we have found it.
+
===(4.4) Interpretation===
 +
</div>
  
This may be due to insufficient resolution of the trees. Whenever we have long, evolutionary distances with a few OTUs clustered at the end, we can be reasonbly certain of the branch. When branching events occur at similar times, the order is uncertain and any specific ordering would probably not be well supported in bootstrapping.
 
 
What is striking however is the parallelism between ''Clade 123'' and ''Clade 118''. Apparently branchpoint 117 marks a duplication event of the ancestral Mbp1 gene in the ''Saccharomycotina''. Here our expectation appears to be borne out. And given the uncertainty in the precise ordering between branchpoints 117 and 128, the Clade overall is thus actually quite consistent with the species tree.
 
  
&nbsp;<br><div style="padding: 5px; background: #EEEEEE;">
+
<div style="padding: 5px; background: #FFCC99;">
*Finally, find one example of a gene duplication, below branchpoint 128.
+
;Analysis (2 marks)
 +
* 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 welcome to upload 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.  
 
</div>
 
</div>
  
 
&nbsp;
 
&nbsp;
 
  
 
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
 
<div style="padding: 5px; background: #BDC3DC;  border:solid 1px #AAAAAA;">
  
==(3) Summary of Resources==
+
==(5) Summary of Resources==
 
</div>
 
</div>
 
&nbsp;<br>
 
&nbsp;<br>
  
;Links
+
;Links and background reading
:* [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/restricted/Baldauf_2003_PhylogenyTutorial.pdf '''Review (PDF, restricted)''' Sandra Baldauf: Phylogeny for the Faint of Heart]
 
:* [[Organism_list_2007|Assigned Organisms]]
 
:* [http://evolution.genetics.washington.edu/phylip.html '''PHYLIP''' home page]
 
:* [http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html '''PHYLIP''' Web Service at the Institut Pasteur]
 
:*[[Assignment_5_fallback_data|'''Fallback data''']]
 
  
;Sequences
+
:* [http://biochemistry.utoronto.ca/undergraduates/courses/BCH441H/restricted/Peitsch_2002_UseOfModels.pdf '''Review (PDF, restricted)''' Manuel Peitsch on Homology Modeling]
:* [[All_APSES_domains|'''All APSES domains''']] (Do not use ... old sequence names!)
+
:* [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 &amp; Burley, winged-Helix domains]
 +
:* [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>
  
;APSES domain alignment
 
:* [[APSES_domains_MUSCLE_revised|All '''APSES domains - MUSCLE aligned''' and sequence names revised]]
 
  
;Trees
+
;Data
:*[[APSES_domains_reference_tree|'''APSES domains reference tree''']]
 
  
&nbsp;
+
:* [[Homology_modeling_fallback_data|'''Fallback Data page''']] <small> - Refer to this page in case your own efforts fail, or you have insurmountable problems with your input files.</small>
&nbsp;
+
 
 +
 
 +
;Reference sequences and alignments
 +
 
 +
:* [[Reference APSES domain sequences (reference species)|'''Reference APSES domains page''']]
  
<div style="padding: 5px; background: #D3D8E8;  border:solid 1px #AAAAAA;">
 
[End of assignment]
 
</div>
 
  
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_2007@googlegroups.com Course Mailing List]
+
{{Template:Assignment_Footer}}

Latest revision as of 17:51, 1 December 2014

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

 
 


   

Assignment 5 (last: 2011) - Homology modeling

How could the search for ultimate truth have revealed so hideous and visceral-looking an object?
Max Perutz (on his first glimpse of the Hemoglobin structure)

   

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 Vendian period of the Proterozoic era of Precambrian times.

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 (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.

In this assignment you will (1) construct a molecular model of the APSES domain from the Mbp1 orthologue in your assigned species, (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,

For the following, please remember the following terminology:

Target
The protein that you are planning to model.
Template
The protein whose structure you are using as a guide to build the model.
Model
The structure that results from the modeling process. It has the Target sequence and is similar to the Template structure.

 

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.

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 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.

Review the guidelines for preparation and submission of BCH441 assignments.

The due date for the assignment is Monday, December 5. at 12:00 noon.

   

 

Your documentation for the procedures you follow in this assignment will be worth 2 marks - 1 mark for generating the model and 1 mark for the selection/superposition and visualization of protein/DNA complexes.

 


(1) Preparation


(1.1) Template choice and template sequence

The SWISS-MODEL server provides several different options for constructing homology models. The easiest is probably the Automated Mode that requires only a target sequence as input, in this mode the program will automatically choose suitable templates and create an input alignment. I disagree however that that is the best way to use such a service: the reason is that template choice and alignment both may be significantly influenced by biochemical reasoning, and an automated algorithm cannot make the necessary decisions. Should you use a structure of reduced resolution that however has a ligand bound? Should you move an indel from an active site to a loop region even though the sequence similarity score might be less? Questions like that may yield answers that are counter to the best choices an automated algorithm could make. Therefore we will use the Alignment Mode of Swiss-Model in this assignment, choose our own template and upload our own alignment.

Template choice is the first step. 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 I have posted a short summary of 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.


In assignment 2 you have already searched for structures of APSES domains in the PDB. If you need to repeat this:

  • Use the NCBI BLAST interface to identify all PDB files that are clearly homologous to your target APSES domain.
  • In Assignment 2, you have defined the extent of the APSES domain in yeast Mbp1. In Assignment 3, you have aligned reference APSES domains with those you found in your species. In assignment 4 you have confirmed by phylogenetic analysis and Recoprocal Best Match which of these APSES domain sequences is the closest related orthologue to yeast Mbp1. This sequence is the best candidate for having a conserved function similar to yeast Mbp1. Therefore, this sequence is the target for the homology modeling procedure.
  • Defining a template means finding a PDB coordinate set that has sufficient sequence similarity to your target that you can huild a model based on theat template. To find suitable PDB structures, use your target sequence as input for a BLAST search, and select Protein Data Bank proteins(pdb) as the Database you search in. Hits that are homologues are all suitable templates in principle, but some are more suitable than others. Consider how the coordinate sets differ and which features would make each more or less suitable for creating a homology model; comment briefly on
  • sequence similarity to your target
  • size of expected model (= length of alignment)
  • presence or absence of ligands
  • experimental method and quality of the data set

Then choose the template you consider the most suitable and note why you have decided to use this template.


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 defined by CDD) is not Residue 1 of the Mbp1 protein. The first residue of the 1MB1 FASTA file (one of the related PDB strcuctures) 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 ATOM records; whereas the SEQRES records start with MET ... and so on. You need to remember: a sequence number is not absolute, but assigned in a particular context.

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 WhatIf, a crystallography and molecular modeling package that offers many useful tools for coordinate manipulation tasks.

  • Navigate to the Administration sub-menu of the 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 ATOM   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 ...
  • ... listed in the SEQRES records of the coordinate file;
  • ... given in the FASTA sequence for the template, which is provided by the PDB;
  • ... stored in the protein database of the NCBI.
and record your results.
  • Establish how the sequence numbers in the coordinate section of your template(*) correspond to your target sequence numbering.
(*) 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..
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..

   

(1.2) The input alignment

 

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.

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.

In most of the Mbp1 orthologues, we do not observe indels in the APSES domain regions - (and for the ones in which we do see indels, we might suspect that these are actually gene-model errors). 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. To obtain an alignment between the template sequence and the target sequence from your species, proceed as follows.

In Jalview...
  • Load your Jalview project with aligned APSES domain sequences or recreate it from the Mbp1 orthologue in your species and the APSES domains from the Reference APSES domain page that I prepared for Assignment 4. Include the sequence of your template protein and re-align.
  • Delete all sequence you no longer need, i.e. keep only the APSES domains of the target (from your species) and the template (from the PDB) and choose Edit → Remove empty columns. This is your input alignment.
  • Choose File→Output to textbox→FASTA to obtain the aligned sequences. They should both have exactly the same length, i.e. N- or C- termini have to be padded by hyphens if the original sequences had different length. Save the sequences in a text-file.


Using a different MSA program
  • Copy the FASTA formatted sequences of the Mbp1 proteins in the reference species from the Reference APSES domain page.
  • Access e.g. the MSA tools page at the EBI.
  • Paste the Mbp1 sequence set, your target sequence and the template sequence into the input form.
  • Run the alignment and save the output.


By hand

APSES domains are strongly conserved and have few if any indels. You could also simply align by hand.

  • Copy the CLUSTAL formatted reference alignment of the Mbp1 proteins in the reference species from the Reference APSES domain page.
  • Open a new file in a text editor.
  • Paste the Mbp1 sequence set, your target sequence and the template sequence into the file.
  • Align by hand, replace all spaces with hyphens and save the output.


Whatever method you use: the result should be a multiple sequence alignment in multi-FASTA format, that was constructed from a number of supporting sequences and that contains your aligned target and template sequence. This is your input alignment for the homology modeling server.


(2) Homology model

   

(2.1) SwissModel

 

Access the Swissmodel server at http://swissmodel.expasy.org . Navigate to the Alignment Mode page.

 

  • Paste your alignment for target and model into the form field. Refer to the Fallback Data file if you are not sure about the format. Make sure to select the correct option (FASTA) for the alignment input format on the form.
  • 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 it came from. Enter the correct Chain-ID (usually "A", note: upper-case).
If you run into problems, compare your input to the fallback data. It has worked for me, it will work for you. In particular we have seen problems that arise from "special" characters in the FASTA header like the pipe "|" character that the NCBI uses to separate IDs - keep the header short and remove all non-alphanumeric characters to be safe.
  • 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.
  • 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.
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.


(3) Model analysis

   

(3.1) The PDB file

 

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:



  • 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 your model correspond to that region?


(3.2) First visualization

 

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.

 

  • Save your model coordinates to your computer and visualize the structure in VMD. Make an informative (parallel, not cross-eyed!) stereo view that shows the general orientation of the helix-turn-helix motif and the "wing", and paste it into your assignment.

 
 

(4) The DNA ligand

   

(4.1) Finding a similar protein-DNA complex

 

One of the really interesting questions we can discuss with reference to our model is how sequence variation might result in changed DNA recognition sites, and then lead to changed cognate DNA binding sequences. In order to address this, we would need to generate a plausible structural model for how DNA is bound to APSES domains.

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 structurally similar protein-DNA complex?

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.

At the NCBI, 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 Structural Analysis page. As part of its MSD Services, PDBeFold 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.

 

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.

 

APSES domains represent one branch of the tree of helix-turn-helix (HTH) DNA binding modules. (A 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. Superfamilies of such structural domains are compiled in the CATH database. Unfortunately CATH itself does not provide information about whether the structures have been determined as complexes. But we can search the PDB with CATH codes and restrict the results to complexes. Essentially, this should give us a list of all winged helix domains for which the structure of complexes with DNA have been determined. This works as follows:

  • For reference, access CATH domain 1.10.10.10; this is the domain you will use to find protein-DNA complexes.
  • Navigate to the PDB home page and follow the link to Advanced Search
  • In the options menu for "Choose a Query Type" select Structure Features → CATH Classification Browser. A window will open that allows you to navigate down through the CATH tree. You can view the Class/Architecture/Topology names on the CATH page linked above. Click on the triangle icons (not the text) for "Mainly Alpha"→"Orthogonal Bundle"→"ARC repressor mutant, subunit A" then click on the link to "winged helix repressor DNA binding domain". Or, just enter "winged helix" into the search field. This subquery should match more than 500 coordinate entries.
  • Click on the (+) button behind "Add search criteria" to add an additional query. Select the option "Structure Features"→"Macromolecule type". In the option menus that pop up, select "Contains Protein → Yes", "Contains DNA → Yes""Contains RNA → Ignore" "Contains DNA/RNA hybrid → 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 "Submit Query". This query should retrieve more than 90 complexes.
  • Scroll down to the beginning of the list of PDB codes and locate the "Generate reports" menu. Under the heading Custom reports select Image collage. 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 in most of the the structures the recognition helix inserts into the major groove of B-DNA (eg. 1BC8, 1CF7). There is one exception: the structure 1DP7 shows how the human RFX1 protein binds DNA in a non-canonical way, through the beta-strands of the "wing". This is interesting since it suggests there is more than one way for winged helix domains to bind to DNA. We can therefore use structural superposition of your homology model and two of the winged-helix proteins to decide whether the canonical or the non-canonical mode of DNA binding seems to be more plausible for Mbp1 orthologues.

 

  • 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 in your documentation for the procedure I have defined above, but I expect you to have spent enough time on this process to understand the key elements of the PDB's advanced search interface.


(4.2) Preparation and superposition of a canonical complex

 

The structure we shall use as a reference for the canonical binding mode is the Elk-1 transcription factor.

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.

The 1DUX coordinate-file contains two protein domains and two B-DNA dimers in one asymmetric unit. For simplicity, you should delete the second copy of the complex from the PDB file. (Remember that PDB files are simply text files that can be edited.)

  • Access the PDB and navigate to the 1DUX structure explorer page. Download the coordinates to your computer.
  • Open the coordinate file in a text-editor and delete the coordinates for chains D,E and F; you may also delete all HETATM records and the MASTER 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 your model's side-chain orientations have not been determined experimentally but inferred from the template, and that the template's structure was determined in the absence of bound DNA ligand.

 

  • 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.

 
 


(4.2) Preparation and superposition of a non-canonical complex


The structure displaying a non-canonical complex between a winged-helix domain and its cognate DNA binding site is the human Regulatory Factor X.

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 coresponds to the recognition 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).


Before we can work with this however, we have to fix an annoying problem caused by the way the PDB stores replicates in biological assemblies. The PDB generates additional chains as copies of the original and delineates them with MODEL and ENDMDL records, just like in a multi-structure NMR file. The chain IDs and the atom numbers are the same as the original. The PDB file thus contains the same molecule in two different orientations, not two independent molecules. This is an important difference regarding how such molecules are displayed by VMD. If you were to use the biological unit file of the PDB, VMD does not recognize that there is a second molecule present and displays only one. We have to edit the file to merge the two molecules by removing the MODEL and ENDMDL records - and while we're editing the file we'll also remove unneeded heteroatoms and the second copy of the protein chain (which we don't need, we need only the second B-DNA strand). But then we end up with residues that have exactly the same residue number in the same file. That won't work for visualization, since the program expects residue numbers to be unique, therefore we have to renumber the residues. Here's how:

  • On the structure explorer page for 1DP7, select the option Download FilesBiological Assembly.
  • Dowload, save and uncompress the file.
  • Open the file in a text editor.
  • Delete both MODEL and both ENDMDL records.
  • Also delete all HETATM records for HOH, PEG and EDO, as well as the entire second protein chain and the MASTER record. The resulting file should only contain the DNA chain and its copy and one protein chain. Save the file with a new name.
  • Access the Whatif Web interface and click on Administration and Renumber a PDB File from 1. Upload your edited file, access the results and save the file.
  • Open the renumbered file with VMD. You should see one protein chain and a B-DNA double helix. Switch to stereo viewing and spend some time to see how amazingly beautiful the complementarity between the protein and the DNA helix is (you might want to display chain P and chain D in separate representations and color the DNA chain by PositionRadial for clarity) ... in particular, appreciate how Arginine 76 interacts with the base of Guanine 92!
  • Then clear all molecules
  • In VMD, open Extensions→Analysis→MultiSeq. 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.
  • 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. Choose File→Import Data, browse to your directory and load:
    • Your model;
    • The 1DUX complex;
    • The 1DP7 complex.
  • Mark all three protein chains by selecting the checkbox next to their name and run the STAMP structural alignment.
  • In the graphical representations window, double-click on the cartoon representations that multiseq has generated to undisplay them, also undisplay the Tube representation of 1DUX. Then create a Tube representation for 1DP7, and select a Color by ColorID (a different color that 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.

 

  • 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.


(4.3) Coloring by conservation

With the superimposed coordinates, you can begin to get a sense whether either or both binding modes could be appropriate for a protein-DNA complex in your Mbp1 orthologue. But these are geometrical criteria only, and the protein in your species may be flexible enough to adopt a different conformation in a complex, and different again from your model. A more powerful way to analyze such hypothetical complexes is to look at conservation patterns. With VMD, you can import a sequence alignment into the MultiSeq extension and color residies by conservation. The protocol below assumes

  • You have prealigned the reference Mbp1 proteins with your species' Mbp1 orthologue;
  • You have saved the alignment in a CLUSTAL format.

You can use Jalview or any other MSA server to do so. You can even do this by hand - there should be few if any indels and the correct alignment is easy to see.


Load the Mbp1 APSES alignment into MultiSeq.
(A) In the MultiSeq Window, navigate to File → 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 the data, it will take a short while to load. If the data can't be loaded, the file may have the wrong extension: .aln 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 N-terminus the sequence that corresponds to the PDB structure has extra residues, and in the middle the APSES sequences may have gaps inserted.

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 → Enable Editing... → 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: S I M ...
(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. (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.)
(E) When you are done, it may be prudent to save the state of your alignment. Use File → Save Session...
Color by similarity
(A) Use the View → Coloring → Sequence similarity → 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 → Graphics → Colors..., choosing the Color Scale tab and adjusting the scale midpoint.
(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.

 

  • Once you have colored the residues of your model by conservation, create another informative stereo-image and paste it into your assignment.


(4.4) Interpretation


Analysis (2 marks)
  • 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 welcome to upload 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.


(5) Summary of Resources

 

Links and background reading


Data
  • Fallback Data page - Refer to this page in case your own efforts fail, or you have insurmountable problems with your input files.


Reference sequences and alignments


[End of assignment]

If you have any questions at all, don't hesitate to mail me at boris.steipe@utoronto.ca or post your question to the 2011 Course Mailing List .