BIN-SX-Homology modelling

Homology Modeling

 

Keywords:  Homology modeling: alignment, alignment, alignment.

 

 

Caution!

This unit is under development. There is some contents here but it is incomplete and/or may change significantly: links may lead to nowhere, the contents is likely going to be rearranged, and objectives, deliverables etc. may be incomplete or missing. Do not work with this material until it is updated to "live" status.

 

Abstract

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This unit ...

Prerequisites

You need to complete the following units before beginning this one:

• BIN-ALI-MSA (Multile Sequence Alignment)
• BIN-SX-Analysis (Analysis of Protein Structure)
• BIN-SX-Domains (Structure domains)
• BIN-SX-Prediction_concepts (Concepts of Structure Prediction)
• BIN-SX-Stereo_vision (Stereo Vision)
• BIN-SX-Superposition (Structure Superposition)

 

Objectives

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Outcomes

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Deliverables

• Time management: Before you begin, estimate how long it will take you to complete this unit. Then, record in your course journal: the number of hours you estimated, the number of hours you worked on the unit, and the amount of time that passed between start and completion of this unit.
• Journal: Document your progress in your Course Journal. Some tasks may ask you to include specific items in your journal. Don't overlook these.
• Insights: If you find something particularly noteworthy about this unit, make a note in your insights! page.

 

Evaluation

Evaluation: NA

This unit is not evaluated for course marks.

 

Contents

Task:

• Read the introductory notes on predicting protein 3D structure by homology modeling.

• Read:

Bienert et al. (2017) The SWISS-MODEL Repository-new features and functionality. Nucleic Acids Res 45:D313-D319. (pmid: 27899672)

[ PubMed ] [ DOI ] SWISS-MODEL Repository (SMR) is a database of annotated 3D protein structure models generated by the automated SWISS-MODEL homology modeling pipeline. It currently holds >400 000 high quality models covering almost 20% of Swiss-Prot/UniProtKB entries. In this manuscript, we provide an update of features and functionalities which have been implemented recently. We address improvements in target coverage, model quality estimates, functional annotations and improved in-page visualization. We also introduce a new update concept which includes regular updates of an expanded set of core organism models and UniProtKB-based targets, complemented by user-driven on-demand update of individual models. With the new release of the modeling pipeline, SMR has implemented a REST-API and adopted an open licencing model for accessing model coordinates, thus enabling bulk download for groups of targets fostering re-use of models in other contexts. SMR can be accessed at https://swissmodel.expasy.org/repository.

 

Introduction

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, 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 RBM orthologue in your assigned species.

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 modelling 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

• We need to define our Target sequence;
• find a suitable structural Template; and
• build a Model.

Target sequence

We have encountered the PDB 1BM8 structure before, the APSES domain of saccharomyces cerevisiae Mbp1. This is a useful template to model the DNA binding domain of your RBM match. But what exactly is the aligned region of the APSES domain? We could use several approaches to define the APSES domain:

• we could use the biostrings package to calculate a pairwise sequence alignment with the 1BM8 sequence, like we did previously for the full-length sequences. This would give us the domain boundaries.
• we could calculate a multiple sequence alignment, while including the 1BM8 sequence. This would also allow us to infer domain boundaries, actually in all sequences in our database at once. But we have found previously that such multiple sequence alignments are quite sensitive to un-alignable regions of which we have quite a few in the full length sequences. We do need an MSA, but we do need to restrict the length of the sequences we align to a reasonable region.
• we could access the domain annotations at CDD or at the SMART Database, but both have interfaces that are difficult to use computationally, and have other issues: NCBI does not recognize APSES domains, only the smaller KilA-N domain, and SMART sometimes does not find APSES domains in our sequences.
• the most straightforward approach of course is to use the annotation that you already have produced for the APSES domain in MBP1_<YFO>. You should be able to simply take the MBP1_SACCE sequence and the one for YFO from the APSES.mfa file.

This is the 1BM8 sequence:

>SACCE
QIYSARYSGVDVYEFIHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRI
LEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAEKFSVYDQLKPLFDF

Template choice and template sequence

The SWISS-MODEL server provides several different options for constructing homology models. The easiest option requires only a target sequence as input. In this mode the program will automatically choose suitable templates and create an input alignment. I would argue however that that is not the best way to use such a service: 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 different from the best choices an automated algorithm could make. But Swiss Model is flexible and allows us to upload an explicit alignment between target and template. Please note: the model you will produce is "easy" - the sequence similarity is high and there are no indels to consider, the automated mode would have done just as well. But the strategy we pursue here is suitable also for much more difficult problems. The automated strategy probably is not.

Template choice is the first step. Often more than one related structure can be found in the PDB. The degree of sequence identity is the most important criterion, but there are many other factors to consider. Please refer to the template choice principles page on this Wiki where I discuss more details and alternatives. To find related structures, you can 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 the BLAST search is probably the method of choice: after all, the most important measure of the probability of success for homology modelling is sequence similarity.

Defining a template means finding a PDB coordinate set that has sufficient sequence similarity to your target that you can build a model based on that template. To find suitable PDB structures, we will perform a BLAST search at the PDB.

Task:

1. Retrieve your aligned YFO's Mbp1 RBM APSES domain sequence from the APSES.mfa selection you have prepared for the phylogeny assignment. This YFO sequence is your target sequence.
2. Navigate to the PDB.
3. Click on Advanced to enter the advanced search interface.
4. Open the menu to Choose a Query Type:
5. Find the Sequence features section and choose Sequence (BLAST...)
6. Paste your target sequence into the Sequence field, select not to mask low-complexity regions and Submit Query. Since the E-value is set rather high by default, you will get a number of low-confidence hits as well as the actual homologs, these have very low E-values.

All hits that are homologs are potentially suitable templates, 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: you should consider ...

• 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

Sequence similarity is the most important, but we can have the PDB tabulate the other features concisely for this task.

1. There is a menu to create Reports: - select customizable table.
2. Select (at least) the following information items:

Structure Summary

• Experimental Method

Sequence

• Chain Length

Ligands

• Ligand Name

Biological details

• Macromolecule Name

refinement Details

• Resolution
• R Work
• R free

1. click: Create report.

Unfortunately you don't get the E-values into the report, and those should strongly influence your final decision. However in our case the sequences and therefore the E-values of the top three hits are all the same. And there is a new structure from January 2015, with a lower resolution. Some of the sequences have a longer chain-length ... but those are only disordered residues (otherwise these would be better suited templates; regrettably, you'd need to check that in the real world, there is no automatic tool to evaluate disorder and its effects on template choice). In my opinion that leaves pretty much only one unambiguous choice for our template: 1BM8.

Finally

Click on the 1BM8 ID to navigate to the structure page for the template and save the FASTA sequence to your computer. This is the template sequence.

 

Sequence numbering

 

It is not straightforward at all how to number sequence in such a project. A "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 1BM8 FASTA file (one of the related PDB structures) is the fourth residue of the Mbp1 protein. The first residue in the structure is GLN 3, therefore Q is the first residue in a FASTA sequence derived from the cordinate section of the PDB file (the ATOM records. In the 1MB1 structure, the original N-terminal amino acids are present in the molecule, therefore they are present in the FASTA file which starts with MSNQIY..., but they are disordered in the structure and no coordinates are present for M and S. A sequence derived explicitly from the coordinates is therefore different from the reported FASTA sequence, which is really bad because that is what the modeling program has to work with ... and so on. It can get complicated. You need to remember: a sequence number is not absolute, but assigned in a particular context and you need to be careful how to do this.

Fortunately, the numbering for the residues in the coordinate section of our target structure corresponds not to its FASTA sequence, but to the numbering of the gene. Otherwise we would need to renumber the sequence (e.g. by using the bio3D R package). If we would not do this, the sequence numbers in the model might not correspond to the sequence numbers of our target.

 

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 just 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. 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.

 

Task:
Choose one of the following options to align your target and template sequence. Make sure your template sequence is included, i.e. the FASTA sequence of 1BM8.

In Jalview...

• Load your APSES domain sequences plus the 1BM8 sequence in Jalview. Include the sequence of your template protein and align using Muscle.
• 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 the MSA tools page at the EBI.
• Paste the Mbp1 sequence set, your target sequence and the template sequence into the input form.
• Run an alignment (I like T-coffee) and save the output.

Using the R bioconductor MSA package that you used previously.

Refer back to the page if you are lacking notes how to go about this.

Whatever method you use: the result should be a two 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. For a Schizosaccharomyces pombe model, which I am using as an example here, it looks like this:

>1BM8_A
QIYSARYSGVDVYEFIHSTGSIMKRKKDDWVNATHILKAANFAKAKRTRI
LEKEVLKETHEKVQGGFGKYQGTWVPLNIAKQLAEKFSVYDQLKPLFDF
>Mbp1_SCHPO 2-100 NP_593032
AVHVAVYSGVEVYECFIKGVSVMRRRRDSWLNATQILKVADFDKPQRTRV
LERQVQIGAHEKVQGGYGKYQGTWVPFQRGVDLATKYKVDGIMSPILSL

In this case, there are no indels and therefore no hyphens - in your case there may be.

 

Homology model

The alignment defines the residue by residue relationship between target and template sequence. All we need to do now is to change every residue of the template to the target sequence

SwissModel

 

Access the Swissmodel server at http://swissmodel.expasy.org and click on the Start Modelling button. Under the Supported Inputs, choose Target-Template Alignment.

Task:

• Paste the aligned sequences of the YFO target and the 1BM8 template into the form field. SwissModel will analyse the sequences and ask you to identify target and template. The YFO sequence is your target. The 1BM8 sequence is the template.

• Click Validate Target Template Alignment and check that the returned alignment is correct. All non-identical residues are shown in light-grey.

• Click Build Model to start the modeling process. This will take about a minute or so.

• The resulting page returns information about the resulting model and its quality. You can rotate the model in the window on the right with the mouse. Regions that have a reddish hue have lower quality scores, i.e. they were harder to model or could not be modelled well with good geometry. Hovering the mouse over parts of the structure highlights the respective region of the sequence alignment.

• Mouse over the Model 01 dropdown menu (under the icon of the template structure), and choose the PDB file. Note that the B-factor column of the coordinate section contains the QMEAN scores (between 0 and 1) that the server has calculated. Higher is better. Save the PDB file on your computer.

• Open the SwissModel documentation in a new tab. Read about the modelling process. there are a number of important technical details that help to understand what the computed coordinates of your model mean, you should pay special attention to the GQME and QMEAN quality scores.

• Also save:

• • The output page as pdf (for reference)
  • The modeling report (as pdf)

Model interpretation

We have spent a significant amount of time to prepare data for the analysis and in practice it usually seems to turn out that way, that the preparation of data occupies the greatest part of our efforts. The actual computational analysis is generally quite fast. And, unfortunately, the interpretation of results is often somewhat neglected. Don't be that way. Data does not explain itself. The interpreattion of your computational results is the most important part.

We will look at our homology model with two different questions:

• Can we define the DNA binding residues?
• Can we tell which residues are conserved for functional reasons, rather than for structural reasons?

   

The PDB file

 

Task:
Open your model coordinates in a text-editor (make sure you view the PDB file in a fixed-width font (like "courier") so all the columns line up correctly) 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?

That's not easy to tell. But it should be.

R code: renumbering the model

As you have seen above, SwissModel numbers the first residue "1" and does not keep the numbering of the template. We should renumber the model so we can compare the model and the template with the same residue numbers. (An alternative renumbering would renumber the model correspond to the sequence it came from. Remember that we have only excised a domain from the full-length sequence.) Carefully doing this by hand will take you a bit less than an hour. Fortunately there is a very useful R package that will help: bio3d.

Task:

1. Navigate to the bio3D home page to . bio3d has recently been made available via CRAN - previously it had to be compiled from source.

1. Explore and execute the following R script. I am assuming that your model is in your PROJECTDIR folder, change paths and filenames as required.

setwd(PROJECTDIR)
PDB_INFILE      <- "YFOmodel.pdb"
PDB_OUTFILE     <- "YFOmodelRenumbered.pdb"


# The bio3d package provides functions for working with
# protein structures in R
if (!require(bio3d, quietly=TRUE)) {
	install.packages("bio3d")
	library(bio3d)
}

# == Read the YFO pdb file

iFirst <- 4  # residue number for the first residue

YFOmodel <- read.pdb(PDB_INFILE) # read the PDB file into a list

YFOmodel           # examine the information
YFOmodel$atom[1,]  # get information for the first atom

# Explore ?read.pdb and study the examples.

# == Modify residue numbers for each atom
resNum <- as.numeric(YFOmodel $atom[,"resno"])
resNum
resNum <- resNum - resNum[1] + iFirst  # add offset
YFOmodel $atom[ , "resno"] <- resNum   # replace old numbers with new

# check result
YFOmodel $atom[ , "resno"]
YFOmodel $atom[1, ]

# == Write output to file
write.pdb(pdb = YFOmodel, file=PDBout)

# Done. Open the PDB file you have written in a text editor
# and confirm that this has worked.

 

First visualization

 

Since a homology model inherits its structural details from the template, your model of the YFO sequence should look very similar to the original 1BM8 structure.

Task:

1. Start Chimera and load the model coordinates that you have just renumbered.
2. From the PDB, also load the template structure. (Use File → Fetch by ID ...)
3. In the Favourites → Model Panel window you can switch between the two molecules.
4. Hide the ribbon and choose backbone only → full. You will note that the backbone of the two structures is virtually identical.
5. Next, choose Actions → Atoms/Bonds → show to display display the two molecules in a stick style and note how the sidechains have been modeled. Note especially how sidechain coordinates have been guessed, where the template had shorter sidechains than the target. It may be more clear if you hide H-atoms: Select → Chemistry → Element → H and Actions → Atoms/Bonds → hide
6. Display only residue 50 to 74 to focus on the putative helix-turn-helix domain. You can drag your mouse in the Favourites → Sequence, window to select the range then Select → Invert (selected model) and Actions → Atoms/Bonds → hide. Or you can use Chimera's commandline: ~display to undisplay everything, show #:50-74 to show this residue range for all models.
7. Study the result: a model of the HTH subdomain of YFO's RBM to Mbp1.

 

Coloring the model by energy

SwissModel calculates energies for each residue of the model with a molecular mechanics forcefield. The SwissModel modeling summary page contains a plot of these energies as a function of sequence number like. The values - between 0.0 and 1.0 - are stored in the PDB file's B-factor field.

Task:

1. Back in Chimera, use the model panel to close the 1BM8 structure. Select all and show Atoms, bonds to view the entire model structure.
2. Choose Tools → Depiction → Render by attribute and select attributes of atoms, Attribute: bfactor, check color atoms and click OK.
3. Study the result: It seems that residues in the core of the protein have better energies (higher values) than residues at the surface. Why could that be the case?

Study the options of this window a bit, rendering by attribute is a powerful way to store and depict all manners of information with the molecule. You can simply write a little R script that uses bio3D to replace the B-factor or occupancy values with any value you might be interested in: energies, conservation scores, information ... whatever. Then render this property to map it on the 3D structure of your molecule...

 

 

Modelling DNA binding

One of the really interesting questions we can discuss with reference to our homology 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 reliably model such a complex from first principles, we will base a model of a bound complex on homology modelling 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. It so happens that early in 2015 an APSES domain structure with bound DNA was published. You probably noticed it as a result of the PDB BLAST search: 4UX5, from the Magnaporthe oryzae Mbp1 orhologue PCG2.

A homologous protein/DNA complex structure

Task:

The PCG2 / DNA complex

• Open Chimera and load the 4UX5 structure. Spend some time exploring it. There are two domains of identical sequence bound to one DNA molecule. The first question I would have is whether the two molecules bind to the same DNA motif - the CGCG core of the "MCB-box", and whether the observed protein:DNA interfaces are actually with the cognate sequence, or whether one (or both) proteins are non-specific complexes. The conditions under which proteins crystallize can be harsh, and physiological function under these conditions is not guaranteed. Indeed, Liu et al. (2015) report that at low concentrations a 1:1 complex is formed and the 2:1 Protein:DNA complex only forms at high concentrations. Figure 3. of their paper shows that the detailed contacts between protein and DNA are in fact not identical.

• Without taking this question too far, let's get a quick view of the comparison by duplicating one domain of the structure and superimposing it on the other. The authors feel that chain A represents the tighter, more specific mode of interaction; so we will duplicate chain B and superpose the copy on A.

• In Chimera, open the Favorites → Model Panel and use the copy/combine button to create a copy of the 4UX5 model. Call it test.
• Select chain B of the test model, then use Select → Invert (selected models) to apply the selection to everything in the test model except chain B.
• Use Actions → Atoms/Bonds → delete to remove everything but Chain B.
• Select and colour the chain red.
• Back on the Model Panel, select both models and use the match... dialogue to open a MatchMaker dialogue window. Choose the radio button two match two specific chains and select 4UX5 chain A as the Reference chain, test chain B as the Chain to match. Click Apply.

You will see that the superimposed structures are very similar, that the main difference is in the orientation of the disordered C-terminus, but also that there is a structural difference between the two structures around Gly 84 which inserts into the minor groove of the double helix.

• Select one of the residues of that loop in chain A by <control>-clicking on it and use Action → Set pivot to set the centre of rotation to that residue: this makes it easier to visualize the binding situation when you make the molecules larger.

• Select residues 81 to 87 and the corresponding (sequence VQGGYGKY) and in both chains turn their ribbon display off and display this range as "sticks".
• Select nucleic acid in the structure submenu and turn ribbons and nucleotide objects off to display the DNA as sticks as well. Colour the DNA by element.
• Study the situation. Focus on Gly 84.A, especially the interaction of its carbonyl oxygen, which hydrogen bonds to the N2 atom of G8.D chain. Gln 89.A hydrogen bonds to the N2 atom of G8.C chain. Gly 84 and Gln 82 thus recognize a G:C C:G pair. In the B chain, Gly 84.B does not contact the DNA well, since it contacts residues of chain A, especially Gln 82.A. The carbonyl atom of Gly 84.B hydrogen bonds to Gln 89.B. and therefore Gln89.B is not available to contact nucleotide bases. What do you think? It seems to me that a crucial interaction for the cognate sequence is contributed by Guanine 8,
• Finally, use the Model Panel to select test and close it.

 

Superimposing your model

Both your homology model and the template structure provide valuable information:

• The template structure shows how conserved the structure is at the protein/DNA interface. You have seen what subtle differences can give rise to a sequence specific complex and a non-specific binding mode. For Mbp1 we know that the APSES domain binds to the same cognate DNA sequence as PCG2. Since your model structure is heavily biased towards the template, evaluating the template in the context of a real protein/DNA complex allows you to judge which binding residues appear to be conserved and possibly modelled in an orientation that is productive for binding.

• The model structure maps sequence variation into that context: are the crucial residues for sequence specific binding conserved?

Task:

• Start by loading your model and the 1BM8 structure into your chimera session. Select all, turn all ribbons off, and set all atoms to stick representation. Then select H atoms by element and hide them.

• We need to visualize and evaluate differences in binding between different proteins and for me it works well to colour everything by element, and give the carbon atoms some identifying, distinct colour. This is best achieved through the Chimera command line that you can turn on with the little "computer" icon on the left-hand side of the graphics window. Have a look at the Chimera Users guide, and choose select to learn how Chimera's selection syntax works.
• Open the Model Panel to check which protein has which Chimera-internal model number. Then you can use the following selection syntax. Instead of the model numbers, I will type <YFO>, <4ux5>, and <1BM8> - you will certainly know by now that these are placeholder labels and you need to replace them with the numbers 0, 1, and 2 instead.

• To colour the DNA carbon atoms white, type:
  

color white #<4ux5>:.C,.D & C

• To colour the 4ux5 A chain carbon atoms grey, type:
  

color #878795 #<4ux5>:.A & C Note: the color values after the first hash are rgb triplets in the hexadecimal numbering systems - exactly like in R.

• To undisplay the 4ux5 B chain, type:
  

~display #<4ux5>:.B Note: this is the tilde character, not a hyphen or minus sign.

• To colour the YFO model carbon atoms a pale reddish color, type:
  

color #b06268 #<YFO> & C

• To colour the 1BM8 structure carbon atoms a pale greenish color, type:
  

color #92b098 #<1BM8> & C

• Ready? Let's superimpose the chains.
  • Select all models in the Model Panel and click on match.
  • Set 4ux5 Chain A as the Reference chain.
  • Select YFO as a Chain to match, select the button for specific reference and specific match, and click Apply.
  • Repeat this with 1BM8 as the match chain.

• Easy. Now enlarge the binding site. Remember that 4ux5 and 1bm8 are independently determined crystal structures, wheres YFO was modelled on 1bm8 and is expected to be very similar to it. To give you some guidance what you should focus on, select 4ux5 residue 84 CA atom and display it as Ball & Stick. You can also repeat the Action "Set Pivot in case the pivot has shifted.

• Study the scene. This is where stereo- vision will help a lot.

• What do you think? Is this what you expected? Can you explain what you see? Was the modelling process succesful?

• Now turn the display of 4ux5 chain B back on and turn chain A off instead. Then superimpose the 1BM8 template and your model on Chain B.

• Again, focus on the binding region. What do you think of that? What would you have expected? Do you see a difference? What does this all mean?

Nb. I haven't seen this before and I am completely intrigued by the results. In fact, I think I understand the protein much, much better now through this exercise. I'm very pleased how this turned out.

Modelling the Ankyrin Domain Section

Creating an Ankyrin domain alignment

APSES domains are relatively easy to identify and annotate but we have had problems with the ankyrin domains in Mbp1 homologues. Both CDD as well as SMART have identified such domains, but while the domain model was based on the same Pfam profile for both, and both annotated approximately the same regions, the details of the alignments and the extent of the predicted region was different.

Mbp1 forms heterodimeric complexes with a homologue, Swi6. Swi6 does not have an APSES domain, thus it does not bind DNA. But it is similar to Mbp1 in the region spanning the ankyrin domains and in 1999 Foord et al. published its crystal structure (1SW6). This structure is a good model for Ankyrin repeats in Mbp1. For details, please refer to the consolidated Mbp1 annotation page I have prepared.

In what follows, we will use the program JALVIEW - a Java based multiple sequence alignment editor to load and align sequences and to consider structural similarity between yeast Mbp1 and its closest homologue in your organism.

In this part of the assignment,

1. You will load sequences that are most similar to Mbp1 into an MSA editor;
2. You will add sequences of ankyrin domain models;
3. You will perform a multiple sequence alignment;
4. You will try to improve the alignment manually;

Jalview, loading sequences

Geoff Barton's lab in Dundee has developed an integrated MSA editor and sequence annotation workbench with a number of very useful functions. It is written in Java and should run on Mac, Linux and Windows platforms without modifications.

Waterhouse et al. (2009) Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189-91. (pmid: 19151095)

[ PubMed ] [ DOI ] UNLABELLED: Jalview Version 2 is a system for interactive WYSIWYG editing, analysis and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightweight Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. AVAILABILITY: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org.

We will use this tool for this assignment and explore its features as we go along.

Task:

1. Navigate to the Jalview homepage click on Download, install Jalview on your computer and start it. A number of windows that showcase the program's abilities will load, you can close these.
2. Prepare homologous Mbp1 sequences for alignment:
   1. Open the Reference Mbp1 orthologues (all fungi) page. (This is the list of Mbp1 orthologs I mentioned above.)
   2. Copy the FASTA sequences of the reference proteins, paste them into a text file (TextEdit on the Mac, Notepad on Windows) and save the file; you could give it an extension of .fa–but you don't have to.
   3. Check whether the sequence for YFO is included in the list. If it is, fine. If it is not, retrieve it from NCBI, paste it into the file and edit the header like the other sequences. If the wrong sequence from YFO is included, replace it and let me know.
3. Return to Jalview and select File → Input Alignment → from File and open your file. A window with sequences should appear.
4. Copy the sequences for ankyrin domain models (below), click on the Jalview window, select File → Add sequences → from Textbox and paste them into the Jalview textbox. Paste two separate copies of the CD00204 consensus sequence and one copy of 1SW6.
   1. When all the sequences are present, click on Add.

Jalview now displays all the sequences, but of course this is not yet an alignment.

Ankyrin domain models

>CD00204 ankyrin repeat consensus sequence from CDD
NARDEDGRTPLHLAASNGHLEVVKLLLENGADVNAKDNDGRTPLHLAAKNGHLEIVKLLL
EKGADVNARDKDGNTPLHLAARNGNLDVVKLLLKHGADVNARDKDGRTPLHLAAKNGHL

>1SW6 from PDB - unstructured loops replaced with xxxx
GPIITFTHDLTSDFLSSPLKIMKALPSPVVNDNEQKMKLEAFLQRLLFxxxxSFDSLLQE
VNDAFPNTQLNLNIPVDEHGNTPLHWLTSIANLELVKHLVKHGSNRLYGDNMGESCLVKA
VKSVNNYDSGTFEALLDYLYPCLILEDSMNRTILHHIIITSGMTGCSAAAKYYLDILMGW
IVKKQNRPIQSGxxxxDSILENLDLKWIIANMLNAQDSNGDTCLNIAARLGNISIVDALL
DYGADPFIANKSGLRPVDFGAG

Computing alignments

try two MSA's algorithms and load them in Jalview.
Locally: which one do you prefer? Modify the consensus. Annotate domains.

The EBI has a very convenient page to access a number of MSA algorithms. This is especially convenient when you want to compare, e.g. T-Coffee and Muscle and MAFFT results to see which regions of your alignment are robust. You could use any of these tools, just paste your sequences into a Webform, download the results and load into Jalview. Easy.

But even easier is to calculate the alignments directly from Jalview. available. (Not today. Bummer.)

No. Calculate an external alignment and import.

Calculate a MAFFT alignment using the Jalview Web service option

Task:

1. In Jalview, select Web Service → Alignment → MAFFT with defaults.... The alignment is calculated in a few minutes and displayed in a new window.

Calculate a MAFFT alignment when the Jalview Web service is NOT available

Task:

1. In Jalview, select File → Output to Textbox → FASTA
2. Copy the sequences.
3. Navigate to the MAFFT Input form at the EBI.
4. Paste your sequences into the form.
5. Click on Submit.
6. Close the Jalview sequence window and either save your MAFFT alignment to file and load in Jalview, or simply 'File → Input Alignment → from Textbox, paste and click New Window.

In any case, you should now have an alignment.

Task:

1. Choose Colour → Hydrophobicity and → by Conservation. Then adjust the slider left or right to see which columns are highly conserved. You will notice that the Swi6 sequence that was supposed to align only to the ankyrin domains was in fact aligned to other parts of the sequence as well. This is one part of the MSA that we will have to correct manually and a common problem when aligning sequences of different lengths.

 

Editing ankyrin domain alignments

A good MSA comprises only columns of residues that play similar roles in the proteins' mechanism and/or that evolve in a comparable structural context. Since the alignment reflects the result of biological selection and conservation, it has relatively few indels and the indels it has are usually not placed into elements of secondary structure or into functional motifs. The contiguous features annotated for Mbp1 are expected to be left intact by a good alignment.

A poor MSA has many errors in its columns; these contain residues that actually have different functions or structural roles, even though they may look similar according to a (pairwise!) scoring matrix. A poor MSA also may have introduced indels in biologically irrelevant positions, to maximize spurious sequence similarities. Some of the features annotated for Mbp1 will be disrupted in a poor alignment and residues that are conserved may be placed into different columns.

Often errors or inconsistencies are easy to spot, and manually editing an MSA is not generally frowned upon, even though this is not a strictly objective procedure. The main goal of manual editing is to make an alignment biologically more plausible. Most comonly this means to mimize the number of rare evolutionary events that the alignment suggests and/or to emphasize conservation of known functional motifs. Here are some examples for what one might aim for in manually editing an alignment:

Reduce number of indels

From a Probcons alignment:
0447_DEBHA    ILKTE-K-T---K--SVVK      ILKTE----KTK---SVVK
9978_GIBZE    MLGLN-PGLKEIT--HSIT      MLGLNPGLKEIT---HSIT
1513_CANAL    ILKTE-K-I---K--NVVK      ILKTE----KIK---NVVK
6132_SCHPO    ELDDI-I-ESGDY--ENVD      ELDDI-IESGDY---ENVD
1244_ASPFU    ----N-PGLREIC--HSIT  ->  ----NPGLREIC---HSIT
0925_USTMA    LVKTC-PALDPHI--TKLK      LVKTCPALDPHI---TKLK
2599_ASPTE    VLDAN-PGLREIS--HSIT      VLDANPGLREIS---HSIT
9773_DEBHA    LLESTPKQYHQHI--KRIR      LLESTPKQYHQHI--KRIR
0918_CANAL    LLESTPKEYQQYI--KRIR      LLESTPKEYQQYI--KRIR

Gaps marked in red were moved. The sequence similarity in the alignment does not change considerably, however the total number of indels in this excerpt is reduced to 13 from the original 22

Move indels to more plausible position

From a CLUSTAL alignment:
4966_CANGL     MKHEKVQ------GGYGRFQ---GTW      MKHEKVQ------GGYGRFQ---GTW
1513_CANAL     KIKNVVK------VGSMNLK---GVW      KIKNVVK------VGSMNLK---GVW
6132_SCHPO     VDSKHP-----------QID---GVW  ->  VDSKHPQ-----------ID---GVW
1244_ASPFU     EICHSIT------GGALAAQ---GYW      EICHSIT------GGALAAQ---GYW

The two characters marked in red were swapped. This does not change the number of indels but places the "Q" into a a column in which it is more highly conserved (green). Progressive alignments are especially prone to this type of error.

Conserve motifs

From a CLUSTAL alignment:
6166_SCHPO      --DKRVA---GLWVPP      --DKRVA--G-LWVPP
XBP1_SACCE      GGYIKIQ---GTWLPM      GGYIKIQ--G-TWLPM
6355_ASPTE      --DEIAG---NVWISP  ->  ---DEIA--GNVWISP
5262_KLULA      GGYIKIQ---GTWLPY      GGYIKIQ--G-TWLPY

The first of the two residues marked in red is a conserved, solvent exposed hydrophobic residue that may mediate domain interactions. The second residue is the conserved glycine in a beta turn that cannot be mutated without structural disruption. Changing the position of a gap and insertion in one sequence improves the conservation of both motifs.

The Ankyrin domains are quite highly diverged, the boundaries not well defined and not even CDD, SMART and SAS agree on the precise annotations. We expect there to be alignment errors in this region. Nevertheless we would hope that a good alignment would recognize homology in that region and that ideally the required indels would be placed between the secondary structure elements, not in their middle. But judging from the sequence alignment alone, we cannot judge where the secondary structure elements ought to be. You should therefore add the following "sequence" to the alignment; it contains exactly as many characters as the Swi6 sequence above and annotates the secondary structure elements. I have derived it from the 1SW6 structure

>SecStruc 1SW6 E: strand   t: turn   H: helix   _: irregular
_EEE__tt___ttt______EE_____t___HHHHHHHHHHHHHHHH_xxxx_HHHHHHH
HHHH_t_____t_____t____HHHHHHH__tHHHHHHHHH____t___tt____HHHHH
HH__HHHH___HHHHHHHHHHHHHEE_t____HHHHHHHHH__t__HHHHHHHHHHHHHH
HHHHHH__EEE_xxxx_HHHHHt_HHHHHHH______t____HHHHHHHH__HHHHHHHH
H____t____t____HHHH___

1SW6_A at the PDBSum database of structure annotations You can compare the diagram there with this text string.

To proceed:

1. Manually align the Swi6 sequence with yeast Mbp1
2. Bring the Secondary structure annotation into its correct alignment with Swi6
3. Bring both CDD ankyrin profiles into the correct alignment with yeast Mbp1

Proceed along the following steps:

Task:

1. Add the secondary structure annotation to the sequence alignment in Jalview. Copy the annotation, select File → Add sequences → from Textbox and paste the sequence.
2. Select Help → Documentation and read about Editing Alignments, Cursor Mode and Key strokes.
3. Click on the yeast Mbp1 sequence row to select the entire row. Then use the cursor key to move that sequence down, so it is directly above the 1SW6 sequence. Select the row of 1SW6 and use shift/mouse to move the sequence elements and edit the alignment to match yeast Mbp1. Refer to the alignment given in the Mbp1 annotation page for the correct alignment.
4. Align the secondary structure elements with the 1SW6 sequence: Every character of 1SW6 should be matched with either E, t, H, or _. The result should be similar to the Mbp1 annotation page. If you need to insert gaps into all sequences in the alignment, simply drag your mouse over all row headers - movement of sequences is constrained to selected regions, the rest is locked into place to prevent inadvertent misalignments. Remember to save your project from time to time: File → save so you can reload a previous state if anything goes wrong and can't be fixed with Edit → Undo.
5. Finally align the two CD00204 consensus sequences to their correct positions (again, refer to the Mbp1 annotation page).
6. You can now consider the principles stated above and see if you can improve the alignment, for example by moving indels out of regions of secondary structure if that is possible without changing the character of the aligned columns significantly. Select blocks within which to work to leave the remaining alignment unchanged. So that this does not become tedious, you can restrict your editing to one Ankyrin repeat that is structurally defined in Swi6. You may want to open the 1SW6 structure in VMD to define the boundaries of one such repeat. You can copy and paste sections from Jalview into your assignment for documentation or export sections of the alignment to HTML (see the example below).

Editing ankyrin domain alignments - Sample

This sample was created by

1. Editing the alignments as described above;
2. Copying a block of aligned sequence;
3. Pasting it To New Alignment;
4. Colouring the residues by Hydrophobicity and setting the colour saturation according to Conservation;
5. Choosing File → Export Image → HTML and pasting the resulting HTML source into this Wikipage.

10 | 20 | 30 | 40 | MBP1_USTMA/341-368   - - Y G D Q L - - - A D - - - - - - - - - - I L - - - - N F Q D D E G E T P L T M A A R A R S MBP1B_SCHCO/470-498   - R E D G D Y - - - K S - - - - - - - - - - F L - - - - D L Q D E H G D T A L N I A A R V G N MBP1_ASHGO/465-494   F S P Q Y R I - - - E T - - - - - - - - - - L I - - - - N A Q D C K G S T P L H I A A M N R D MBP1_CLALU/550-586   G N Q N G N S N D K K E - - - - - - - - - - L I S K F L N H Q D N E G N T A F H I A A Y N M S MBPA_COPCI/514-542   - H E G G D F - - - R S - - - - - - - - - - L V - - - - D L Q D E H G D T A I N I A A R V G N MBP1_DEBHA/507-550   I R D S Q E I - - - E N K K L S L S D K K E L I A K F I N H Q D I D G N T A F H I V A Y N L N MBP1A_SCHCO/388-415   - - Y P K E L - - - A D - - - - - - - - - - V L - - - - N F Q D E D G E T A L T M A A R C R S MBP1_AJECA/374-403   T L P P H Q I - - - S M - - - - - - - - - - L L - - - - S S Q D S N G D T A A L A A A K N G C MBP1_PARBR/380-409   I L P P H Q I - - - S L - - - - - - - - - - L L - - - - S S Q D S N G D T A A L A A A K N G C MBP1_NEOFI/363-392   T C S Q D E I - - - D L - - - - - - - - - - L L - - - - S C Q D S N G D T A A L V A A R N G A MBP1_ASPNI/365-394   T F S P E E V - - - D L - - - - - - - - - - L L - - - - S C Q D S V G D T A V L V A A R N G V MBP1_UNCRE/377-406   M Y P H H E V - - - G L - - - - - - - - - - L L - - - - A S Q D S N G D T A A L T A A K N G C MBP1_PENCH/439-468   T C S Q D E I - - - Q M - - - - - - - - - - L L - - - - S C Q D Q N G D T A V L V A A R N G A MBPA_TRIVE/407-436   V F P R H E I - - - S L - - - - - - - - - - L L - - - - S S Q D A N G D T A A L T A A K N G C MBP1_PHANO/400-429   T W I P E E V - - - T R - - - - - - - - - - L L - - - - N A Q D Q N G D T A I M I A A R N G A MBPA_SCLSC/294-313   - - - - - - - - - - - - - - - - - - - - - - - L - - - - D A R D I N G N T A I H I A A K N K A MBPA_PYRIS/363-392   T W I P E E V - - - T R - - - - - - - - - - L L - - - - N A A D Q N G D T A I M I A A R N G A MBP1_/361-390   - - - N H S L G V L S Q - - - - - - - - - - F M - - - - D T Q N N E G D T A L H I L A R S G A MBP1_ASPFL/328-364   T E Q P G E V I T L G R - - - - - - - - - - F I S E I V N L R D D Q G D T A L N L A G R A R S MBPA_MAGOR/375-404   Q H D P N F V - - - Q Q - - - - - - - - - - L L - - - - D A Q D N D G N T A V H L A A Q R G S MBP1_CHAGL/361-390   S R S A D E L - - - Q Q - - - - - - - - - - L L - - - - D S Q D N E G N T A V H L A A M R D A MBP1_PODAN/372-401   V R Q P E E V - - - Q A - - - - - - - - - - L L - - - - D A Q D E E G N T A L H L A A R V N A MBP1_LACTH/458-487   F S P R Y R I - - - E N - - - - - - - - - - L I - - - - N A Q D Q N G D T A V H L A A Q N G D MBP1_FILNE/433-460   - - Y P Q E L - - - A D - - - - - - - - - - V I - - - - N F Q D E E G E T A L T I A A R A R S MBP1_KLULA/477-506   F T P Q Y R I - - - D V - - - - - - - - - - L I - - - - N Q Q D N D G N S P L H Y A A T N K D MBP1_SCHST/468-501   A K D P D N K - - - K D - - - - - - - - - - L I A K F I N H Q D S D G N T A F H I C S H N L N MBP1_SACCE/496-525   F S P Q Y R I - - - E L - - - - - - - - - - L L - - - - N T Q D K N G D T A L H I A S K N G D CD00204/1-19   - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A R D E D G R T P L H L A A S N G H CD00204/99-118   - - - - - - - - - - - - - - - - - - - - - - - V - - - - N A R D K D G R T P L H L A A K N G H 1SW6/203-232   L D L K W I I - - - A N - - - - - - - - - - M L - - - - N A Q D S N G D T C L N I A A R L G N SecStruc/203-232   t _ H H H H H - - - H H - - - - - - - - - - _ _ - - - - _ _ _ _ t _ _ _ _ H H H H H H H H _ _

Aligned sequences before editing. The algorithm has placed gaps into the Swi6 helix LKWIIAN and the four-residue gaps before the block of well aligned sequence on the right are poorly supported.

10 | 20 | 30 | 40 | MBP1_USTMA/341-368   - - Y G D Q L A D - - - - - - - - - - - - - - I L N F Q D D E G E T P L T M A A R A R S MBP1B_SCHCO/470-498   - R E D G D Y K S - - - - - - - - - - - - - - F L D L Q D E H G D T A L N I A A R V G N MBP1_ASHGO/465-494   F S P Q Y R I E T - - - - - - - - - - - - - - L I N A Q D C K G S T P L H I A A M N R D MBP1_CLALU/550-586   G N Q N G N S N D K K E - - - - - - - L I S K F L N H Q D N E G N T A F H I A A Y N M S MBPA_COPCI/514-542   - H E G G D F R S - - - - - - - - - - - - - - L V D L Q D E H G D T A I N I A A R V G N MBP1_DEBHA/507-550   I R D S Q E I E N K K L S L S D K K E L I A K F I N H Q D I D G N T A F H I V A Y N L N MBP1A_SCHCO/388-415   - - Y P K E L A D - - - - - - - - - - - - - - V L N F Q D E D G E T A L T M A A R C R S MBP1_AJECA/374-403   T L P P H Q I S M - - - - - - - - - - - - - - L L S S Q D S N G D T A A L A A A K N G C MBP1_PARBR/380-409   I L P P H Q I S L - - - - - - - - - - - - - - L L S S Q D S N G D T A A L A A A K N G C MBP1_NEOFI/363-392   T C S Q D E I D L - - - - - - - - - - - - - - L L S C Q D S N G D T A A L V A A R N G A MBP1_ASPNI/365-394   T F S P E E V D L - - - - - - - - - - - - - - L L S C Q D S V G D T A V L V A A R N G V MBP1_UNCRE/377-406   M Y P H H E V G L - - - - - - - - - - - - - - L L A S Q D S N G D T A A L T A A K N G C MBP1_PENCH/439-468   T C S Q D E I Q M - - - - - - - - - - - - - - L L S C Q D Q N G D T A V L V A A R N G A MBPA_TRIVE/407-436   V F P R H E I S L - - - - - - - - - - - - - - L L S S Q D A N G D T A A L T A A K N G C MBP1_PHANO/400-429   T W I P E E V T R - - - - - - - - - - - - - - L L N A Q D Q N G D T A I M I A A R N G A MBPA_SCLSC/294-313   - - - - - - - - - - - - - - - - - - - - - - - - L D A R D I N G N T A I H I A A K N K A MBPA_PYRIS/363-392   T W I P E E V T R - - - - - - - - - - - - - - L L N A A D Q N G D T A I M I A A R N G A MBP1_/361-390   N H S L G V L S Q - - - - - - - - - - - - - - F M D T Q N N E G D T A L H I L A R S G A MBP1_ASPFL/328-364   T E Q P G E V I T L G R F I S E - - - - - - - I V N L R D D Q G D T A L N L A G R A R S MBPA_MAGOR/375-404   Q H D P N F V Q Q - - - - - - - - - - - - - - L L D A Q D N D G N T A V H L A A Q R G S MBP1_CHAGL/361-390   S R S A D E L Q Q - - - - - - - - - - - - - - L L D S Q D N E G N T A V H L A A M R D A MBP1_PODAN/372-401   V R Q P E E V Q A - - - - - - - - - - - - - - L L D A Q D E E G N T A L H L A A R V N A MBP1_LACTH/458-487   F S P R Y R I E N - - - - - - - - - - - - - - L I N A Q D Q N G D T A V H L A A Q N G D MBP1_FILNE/433-460   - - Y P Q E L A D - - - - - - - - - - - - - - V I N F Q D E E G E T A L T I A A R A R S MBP1_KLULA/477-506   F T P Q Y R I D V - - - - - - - - - - - - - - L I N Q Q D N D G N S P L H Y A A T N K D MBP1_SCHST/468-501   A K D P D N K K D - - - - - - - - - - L I A K F I N H Q D S D G N T A F H I C S H N L N MBP1_SACCE/496-525   F S P Q Y R I E L - - - - - - - - - - - - - - L L N T Q D K N G D T A L H I A S K N G D CD00204/1-19   - - - - - - - - - - - - - - - - - - - - - - - - - N A R D E D G R T P L H L A A S N G H CD00204/99-118   - - - - - - - - - - - - - - - - - - - - - - - - V N A R D K D G R T P L H L A A K N G H 1SW6/203-232   L D L K W I I A N - - - - - - - - - - - - - - M L N A Q D S N G D T C L N I A A R L G N SecStruc/203-232   t _ H H H H H H H - - - - - - - - - - - - - - _ _ _ _ _ _ t _ _ _ _ H H H H H H H H _ _

Aligned sequence after editing. A significant cleanup of the frayed region is possible. Now there is only one insertion event, and it is placed into the loop that connects two helices of the 1SW6 structure.

Final analysis of the ankyrin alignment

Task:

• Compare the distribution of indels in the ankyrin repeat regions of your alignments.
  • Review whether the indels in this region are concentrated in segments that connect the helices, or if they are more or less evenly distributed along the entire region of similarity.
  • Think about whether the assertion that indels should not be placed in elements of secondary structure has merit in your alignment.
  • Recognize that an indel in an element of secondary structure could be interpreted in a number of different ways:
    • The alignment is correct, the annotation is correct too: the indel is tolerated in that particular case, for example by extending the length of an α-helix or β-strand;
    • The alignment algorithm has made an error, the structural annotation is correct: the indel should be moved a few residues;
    • The alignment is correct, the structural annotation is wrong, this is not a secondary structure element after all;
    • Both the algorithm and the annotation are probably wrong, but we have no data to improve the situation.

(NB: remember that the structural annotations have been made for the yeast protein and might have turned out differently for the other proteins...)

You should be able to analyse discrepancies between annotation and expectation in a structured and systematic way. In particular if you notice indels that have been placed into an annotated region of secondary structure, you should be able to comment on whether the location of the indel has strong support from aligned sequence motifs, or whether the indel could possibly be moved into a different location without much loss in alignment quality.

• Considering the whole alignment and your experience with editing, you should be able to state whether the position of indels relative to structural features of the ankyrin domains in your organism's Mbp1 protein is reliable. That would be the result of this task, in which you combine multiple sequence and structural information.

• You can also critically evaluate database information that you have encountered:

1. Navigate to the CDD annotation for yeast Mbp1.
2. You can check the precise alignment boundaries of the ankyrin domains by clicking on the (+) icon to the left of the matching domain definition.
3. Confirm that CDD extends the ankyrin domain annotation beyond the 1SW6 domain boundaries. Given your assessment of conservation in the region beyond the structural annotation: do you think that extending the annotation is reasonable also in YFO's protein? Is there evidence for this in the alignment of the CD00204 consensus with well aligned blocks of sequence beyond the positions that match Swi6?

From Homology Modeling - Modeling alternative binding modes

 

Finding a 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.

Task:

1. Navigate to the VAST search interface page.
2. Enter 1bm8 as the PDB ID to search for and click Go.
3. Follow the link to Related Structures.
4. Study the result.

You will see that VAST finds more than 3,000 partially similar structures, but it would be almost impossibly tedious to manually search through the list for structures of protein DNA complexes that are similar to the interacting core of the APSES domain. It turns out that our search is not specific enough in two ways: we have structural elements in our PDB file that are unnecessary for the question at hand, and thus cause the program to find irrelevant matches. But, if we constrain ourselves to just a single helix and strand (i.e. the 50-74 subdomain that has been implicated in DNA binding, the search will become too non-specific. Also we have no good way to retrieve functional information from these hits: which ones are DNA-binding proteins, that bind DNA through residues of this subdomain and for which the structure of a complex has been solved? It seems we need to define our question more precisely.

Task:

1. Open VMD and load the 1BM8 structure or your YFO homology model.
2. Display the backbone as a Trace (of CA atoms) and color by Index
3. In the sequence viewer, highlight residues 50 to 74.
4. In the representations window, find the yellow representation (with Color ID 4) that the sequence viewer has generated. Change the Drawing Method to NewCartoon.
5. Now (using stereo), study the topology of the region. Focus on the helix at the N-terminus of the highlighted subdomain, it is preceded by a turn and another helix. This first helix makes interactions with the beta hairpin at the C-terminal end of the subdomain and is thus important for the orientation of these elements. (This is what is referred to as a helix-turn-helix motif, or HtH motif, it is very common in DNA-binding proteins.)
6. Holding the shift key in the alignment viewer, extend your selection until you cover all of the first helix, and the residues that contact the beta hairpin. I think that the first residue of interest here is residue 33.
7. Again holding the shift key, extend the selection at the C-terminus to include the residues of the beta hairpin to where they contact the helix at the N-terminus. I think that the last residue of interest here is residue 79.
8. Study the topology and arrangement of this compact subdomain. It contains the DNA-binding elements and probably most of the interactions that establish its three-dimensional shape. This subdomain even has a name: it is a winged helix DNA binding motif, a member of a very large family of DNA-binding domains. I have linked a review by Gajiwala and Burley to the end of this page; note that their definition of a canonical winged helix motif is a bit larger than what we have here, with an additional helix at the N-terminus and a second "wing". )

Armed with this insight, we can attempt again to find meaningfully similar structures. 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 for our purpose

Task:

1. Navigate to the PDBeFold search interface page.
2. Enter 1bm8 for the PDB code and choose Select range from the drop down menu. Select the residues you have defined above.
3. Note that you can enter the lowest acceptable match % separately for query and target. This means: what percentage of secondary structure elements would need to be matched in either query or target to produce a hit. Keep that value at 80 for our query, since we would want to find structures with almost all of the elements of the winged helix motif. Set the match to 10 % for the target, since we are interested in such domains even if they happen to be small subdomains of large proteins.
4. Keep the Precision at normal. Precision and % query match could be relaxed if we wanted to find more structures.
5. Finally click on: Submit your query.
6. On the results page, click on the index number (in the left-hand column) of the top hit that is not one of our familiar Mbp1 structures to get a detailed view of the result. Most likely this is 1wq2:a, an enzyme. Click on View Superposed. This will open a window with the structure coordinates superimposed in the Jmol molecular viewer. Control-click anywhere in the window area to open a menu of viewing options. Select Style → Stereographic → Wall-eyed viewing. Select Trace as the rendering. Then study the superposition. You will note that the secondary structure elements match quite well, but does this mean we have a DNA-binding domain in this sulfite reductase?

All in all this appears to be well engineered software! It gives you many options to access result details for further processing. I think this can be put to very good use. But for our problem, we would have to search through too many structures because, once again, we can't tell which ones of the hits are DNA binding domains, especially domains for which the structure of a complex has 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:

Task:

• For reference, access CATH domain superfamily 1.10.10.10; this is the CATH classification code we will use to find protein-DNA complexes. Click on Superfamily Superposition to get a sense of the structural core of the winged helix domain.

1. Navigate to the PDB home page and follow the link to Advanced Search
2. 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 550 coordinate entries.
3. 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.
4. Check the box below this subquery to Remove Similar Sequences at 90% identity and click on Submit Query. This query should retrieve more than 100 complexes.
5. Scroll down to the beginning of the list of PDB codes and locate the Reports menu. Under the heading View select Gallery. This is a fast way to obtain an overview of the structures that have been returned. Adjust the number of Results to see all 100 images and choose Options→Resize medium.
6. Finally we have a set of winged-helix domain/DNA complexes, for comparison. Scroll through the gallery and study how the protein binds DNA.

First of all you may notice that in fact not all of the structures are really different, despite having requested only to retrieve dissimilar sequences, and not all images show DNA. 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) and the wing - if clearly visible at all in the image - appears to make accessory interactions with the DNA backbone.. 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.

 

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.)

Task:

1. Find the 1DUX structure in the image gallery and open the 1DUX structure explorer page in a separate window. Download the coordinates to your computer.
2. Open the coordinate file in a text-editor (TextEdit or Notepad - NOT MS-Word!) 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 .
3. 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.
4. 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.
5. In the "'Stamp Alignment Options'" window, check the radio-button for Align the following ... Marked Structures and click on OK.
6. In the Graphical Representations window, double-click on all "NewCartoon" representations for both molecules, to undisplay them.
7. 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.

1. 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. You may want to keep a copy of the image for future reference. Consider which parts of the structure appear to superimpose best. Note whether it is plausible that your model could bind a B-DNA double-helix in this orientation.

 
 

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. If you download and view the 1DP7 structure in VMD, you will notice that there is only a single strand of DNA! Where is the second strand of the double helix? It is not in the coordinate file, because it happens to be exactly equivalent to the frist starnd, rotated around a two-fold axis of symmetry in the crystal lattice. We need to download and work with the so-called Biological Assembly instead. But there is a problem related to the way the PDB stores replicates in biological assemblies. The PDB generates the 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 try to use the biological unit file of the PDB, VMD does not recognize that there is a second molecule present and displays only one chain. And that looks exactly like the one we have seen before. We have to edit the file, extract the second DNA molecule, change its chain ID and then append it to the original 1DP7 structure...

Task:

1. On the structure explorer page for 1DP7, select the option Download Files → PDB File.
2. Also select the option Download Files → Biological Assembly.
3. Uncompress the biological assembly file.
4. Open the file in a text editor.
5. Delete everything except the second DNA molecule. This comes after the MODEL 2 line and has chain ID D. Keep the TER and END lines. Save this with a new filename (e.g. 1DP7_DNAonly.pdb).
6. 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, eg. 1DP7_BDNA.PDB.
7. Use a similar procedure as BIO_Assignment_Week_8#R code: renumbering the model in the last assignment to change the chain ID.

PDBin <- "1DP7_DNAonly.pdb"
PDBout <- "1DP7_DNAnewChain.pdb"

pdb  <- read.pdb(PDBin)
pdb$atom[,"chain"] <- "E"
write.pdb(pdb=pdb,file=PDBout)

1. Use your text-editor to open both the 1DP7.pdb structure file and the 1DP7_DNAnewChain.pdb. Copy the DNA coordinates, paste them into the original file before the END line and save.
2. Open the edited coordinate file with VMD. You should see one protein chain and a B-DNA double helix. (Actually, the BDNA helix has a gap, because the R-library did not read the BRDU nucleotide as DNA). 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 protein and nucleic in separate representations and color the DNA chain by Position → Radial for clarity) ... in particular, appreciate how not all positively charged side chains contact the phosphate backbone, but some pnetrate into the helix and make detailed interactions with the nucleobases!
3. Then clear all molecules
4. 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.
5. Choose File→Import Data, browse to your directory and load one by one:

-Your model;

-The 1DUX complex;

-The 1DP7 complex.

1. Mark all three protein chains by selecting the checkbox next to their name and choose Tools→ STAMP structural alignment.
2. Align the Marked Structures, choose a scanscore of 2 and scanslide of 5. Also choose Slow scan. You may have to play around with the setting to get the molecules to superimpose: but the can be superimposed quite well - at least the DNA-binding helices and the wings should line up.
3. 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.
4. Orient and scale your superimposed structures so that their structural similarity is apparent, and the differences in binding elements is clear. Perhaps visualizing a solvent accessible surface of the DNA will help understand the spatial requirements of the complex formation. You may want to keep a copy of the image for future reference. Note whether it is plausible that your model could bind a B-DNA double-helix in the "alternative" conformation.

 

Task:

1. Spend some time studying the complex.
2. Recapitulate in your mind how we have arrived at this comparison, in particular, how this was possible even though the sequence similarity between these proteins is low - none of these winged helix domains came up as a result of our previous BLAST search in the PDB.
3. You should clearly think about the following question: considering the position of the two DNA helices relative to the YFO structural model, which binding mode appears to be more plausible for protein-DNA interactions in the YFO Mbp1 APSES domains? Is it the canonical, or the non-canonical binding mode? Is there evidence that allows you to distinguish between the two modes?
4. Before you quit VMD, save the "state" of your session so you can reload it later. We will look at residue conservation once we have built phylogenetic trees. In the main VMD window, choose File→Save State....

 

Further reading, links and resources

 

Notes

 

Self-evaluation

 

 

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If in doubt, ask! If anything about this learning unit is not clear to you, do not proceed blindly but ask for clarification. Post your question on the course mailing list: others are likely to have similar problems. Or send an email to your instructor.

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About ...
 
Author:

Boris Steipe <boris.steipe@utoronto.ca>

Created:

2017-08-05

Modified:

2017-08-05

Version:

0.1

Version history:

• 0.1 First stub

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