After having recently published a large scale Molecular Dynamics simulations project of TCRpMHC [1,2] interaction I have extended my research to another technique of spatial sampling. At this week’s group meeting I presented the first results of my first MOSAICS [3] project.

The MOSAICS package is a software that allows for so called hierarchical natural Monte Carlo moves. That means that the user can specify regions in the protein of interest. These regions are indented to reflect “natural” sets of atoms and are expected to move together. An example would be a stable alpha-helix. “Hierarchical” means that region can be grouped together to super-regions. For example a helix that is broken by a kink [4] in its middle could have a region for the helix parts on both sides of the kink as well as for the overall helix. An example for peptide/MHC is illustrated below.

MOSAICS uses Monte Carlo moves to rearrange these region with respect to each other. A stochastic chain closure algorithm ensures that no chain breaks occur. An example of such movements in comparison to classical all-atom Molecular Dynamics is shown below.

In this study we used MOSAICS to simulate the detachment of peptides from MHCs for experimentally known binder and non-binder. An example of such a detaching peptide is shown below

Our results show that experimentally known non-binding peptides detach significantly faster from MHC than experimentally known binding peptides (results to be reported soon).

As a first conclusion of this project:
After having worked with both MOSAICS and Molecular Dynamics simulations, I think that both techniques have their advantages and disadvantages. They are summarized below:

Which technique should be chosen for which project depends mainly on what the aims of these projects are. If large moves of well defined segments are expected then MOSAICS might be the method of choice. If the aim is to investigate fine changes and detailed dynamics Molecular Dynamics simulations might be the better choice.

 

1.    Knapp B, Demharter S, Esmaielbeiki R, Deane CM (2015) Current Status and Future Challenges in T-cell receptor / peptide / MHC Molecular Dynamics Simulations. Brief Bioinform accepted.
2.    Knapp B, Dunbar J, Deane CM (2014) Large Scale Characterization of the LC13 TCR and HLA-B8 Structural Landscape in Reaction to 172 Altered Peptide Ligands: A Molecular Dynamics Simulation Study. PLoS Comput Biol 10: e1003748.
3.    Sim AY, Levitt M, Minary P (2012) Modeling and design by hierarchical natural moves. Proc Natl Acad Sci U S A 109: 2890-2895.
4.    Wilman HR, Ebejer JP, Shi J, Deane CM, Knapp B (2014) Crowdsourcing yields a new standard for kinks in protein helices. J Chem Inf Model 54: 2585-2593.

Last week I discussed the recent paper by Lhota et al. proposing a network-based similarity search method, applied to the problem of protein fold prediction. Similarity search is the foundation of bioinformatics. It plays a key role in establishing structural, functional and evolutionary relationships between biological sequences. Although the power of the similarity search has increased steadily in recent years, a high percentage of sequences remain uncharacterized in the protein universe. Cumulative evidence suggests that the protein universe is continuous. As a result, conventional sequence homology search methods may be not able to detect novel structural, functional and evolutionary relationships between proteins from weak and noisy sequence signals. To overcome the limitations in existing similarity search methods, the authors propose a new algorithmic framework, Enrichment of Network Topological Similarity (ENTS). While the method is general in scope, in the paper, authors focus exclusively on the protein fold recognition problem.

To initialize ENTS for structure prediction, ENTS builds a structural similarity graph of protein domains (Fig 1). The structural similarity graph is a weighted graph with one node for each structural domain and an edge between two nodes only if their pairwise similarity exceeds a certain threshold. In this article, the structural similarity score is determined by TM align with a threshold of 0.4. Next, some or all the structural domains in the database are labeled with SCOP. Given a query domain sequence and the goal to predict its structure, ENTS first links the query to all nodes in the structural similarity graph. The weights of these new edges are based only on the sequence profile-profile similarity derived from HHSearch. Then random walk with restart (RWR) is applied to perform a probabilistic traversal of the instance graph across all paths leading away from the query, where the probability of choosing an edge will be proportional to its weight. The algorithm will output a ranked list of probabilities of reaching each node in the structural graph from the query, thus potentially uncovering relationships missed by pair-wise comparison methods. ENTS also uses an enrichment analysis step to assess the reliability of the detected relationships, by comparing the mean relationship strength of a SCOP cluster in the structural graph and the query, to that of random clusters.

For testing the method, the authors first constructed a structural graph using 36,003 non-redundant protein domains from the PDB. The query benchmark set consisted of 885 SCOP domains, constructed by randomly selecting each domain from folds spanning at least two super-families. An additional step before prediction on the query set was to remove all domains from the structral graph which were in the same super-family as the query. The method was compared to existing methods such as CNFPred and HHSearch and it’s network approach and enrichment analysis step were found to contribute significantly to the accuracy of fold prediction. While the method seems to be an improvement on existing methods and is a novel use of network-based approaches to fold prediction, the false positive rate is still very high. One way of overcoming this, suggested by the authos is the use of energy-based scoring functions to further prune the list of potential hits returned by the method.

Let’s assume you have a child. Tom is 5 years old, he’s a piece of work. He loves running around the house and the only time you can get a bit of rest is when he’s drawing. Tom loves drawing, so you use that whenever you need a bit of time to sit down. Today is one of those days, you didn’t sleep well and there was trouble at work. However, when you pick up Tom from daycare, he’s excited and full of energy, so you suggest Tom draw a farm which you can put on the wall in your office at work. You sit down and Tom is busy for half an hour. After half an hour Tom proudly presents his work. There are pigs in sties, horses in stables and cows on the meadow. He looks up at you and asks: “Is this a good farm?”. Of course, you say it is an amazing farm.

Only, what if you didn’t know what a farm looked like? You could ask the neighbour’s kid, Emily, to draw a farm and see what the images have in common? You could do this at a whole different scale and start a drawing competition for all the 5-year-olds in the neighbourhood and look which 5-year-olds draw most accurately to see who you should believe what a farm looks like. Clearly we’re not talking about children drawing farms any more. But what if Tom were a functional similarity metric that has just evaluated the results of a community detection algorithm run on a protein interaction network to generate communities?

That was a bit of a jump there. Let me explain. I have spent the last 2 years looking into how protein interaction networks (pins) can be partitioned into functional biological modules. It is widely believed that functional modules are an important scale of organization in humans or any other organisms (c.f. Hartwell et al 1999). These modules consist of proteins which perform the same or similar biological functions via interacting with each other. Thus, there may be a module which contains proteins that interact to e.g. heal wounds.

 

Finding Functional Modules

We attempt to find these modules by looking at a network which contains data on which proteins interact with which other ones (pins) and then use algorithms to group proteins together based on these interactions. The resulting protein communities contain proteins that interact more with each other than with the rest of the network. So that’s that, right?

…No, sadly it isn’t. The issue is that there are probably tens, if not hundreds of algorithms to find these communities and they don’t agree with each other. Furthermore, the underlying network contains a lot of false interactions and is missing a lot of true interactions. This affects different community detection methods in different ways. So we need a way to evaluate the results these community detection algorithms are presenting. But how do we judge what a good community is when it is exactly this community that we are looking for? How do we know it is “a good farm”, when we don’t know what a farm looks like?

 

Evaluating Community Detection by Functional Similarity

Luckily we do have some idea of what is “good”. Most proteins have annotations which suggest what biological functions they are involved in. As we are looking for functional modules which group together proteins involved in the same or a similar function, this is exactly along our alleyway! Lucky us :). Right?

Again, you might have expected this one, the answer is “not entirely”. Not only are there a lot of ways to find communities in a network, there are also a huge number of ways to use the above-mentioned functional annotations (GO annotations, www.geneontology.org) to calculate how “functionally similar” two proteins are. Now you might think that all these functional similarity measures use the same functional annotation data, so they should generally agree on what is a “good” and what is a “bad” community. This was my first intuition, so I checked. Below are two graphs which show the results of this test. In both cases I evaluate exactly the same sets of communities which I get from partitioning a pin using Link Clustering (Ahn et al 2010) at different resolutions. The plots show the average functional homogeneity of communities in different community size bands as judged by the Pandey Method on the left (Pandey et al 2008), and the simGIC method on the right (Pesquita et al 2007).

You can clearly see that the two plots look different. Now it would be okay if they looked different and still agreed on the most important part: where is my pin partitioned into communities in the best way? To check this I look for maxima in the plots. Figure A) tells me that at a resolution of about 0.2 communities of size 2-35 are on average quite functionally homogeneous. At a slightly lower resolution (approx 0.1) communities of size 36+ look like they are partitioned decently. So depending on the community sizes I’m looking for, I can say which resolution I should go for. However, these peaks don’t show up in Figure B). We clearly need an evaluation of the evaluation metric. Which one should I believe?

Features that are common to both cases (the yellow peak around 0.4 and the magenta maximum around 0.3) might be “true”. But what is to say that the first set of peaks isn’t also important? In our earlier analogy, the neighbour’s daughter Emily has drawn a farm that looks quite different. You’re not about to say Tom’s wrong only because Emily drew a different picture, right? So right about now we need that drawing competition!

 

Evaluating Evaluation metrics: The drawing competition

Now we’ve stumbled twice already in how to evaluate our results. This time, we should make sure that we can evaluate the different results confidently. The plan is that we let the kids draw something else. Not a farm¸ because we don’t know how that looks like. We ask them to draw something related, like a house. We live in a house, so that should be easier to evaluate. And apparently houses and farms are not too dissimilar, so that the kids that do well in their house drawings, may be the ones that have the best farm drawings as well (or so we hope).

In terms of PINs, we have to create a network with community structure which looks a bit like a PIN. We can then use a community detection algorithm to find the communities and let our kids (functional similarity metrics) go away and evaluate the communities at different resolutions. This time we however know which maxima should show up, as we created the network and therefore know the community structure that should be found.

To create a PIN-similar network is not a mean feat and can be the topic of a whole PhD thesis, so I have focussed on a small, simple network, which is hopefully PIN-similar enough to be a meaningful evaluation for the functional similarity metrics. My network is generated on the basis of functional labels ordered in the ontology below.

Each node is assigned one or two labels between 6 and 15 randomly without replacement. Then the ontology is traced upwards to get the ancestral label sets associated with each node (i.e. label 15 has the ancestral labels 5 and 1). Edge probabilities are calculated between two nodes based on the number of common labels these nodes have in their ancestral label sets. Finally edges are added based on the edge probabilities to give a network with a density comparable to a PIN.

When this network is clustered into communities and functionally evaluated at different resolutions, the results look like this:

The black graph shows how many labels nodes in the same community have in common at different resolutions. As these labels overlaps were used to generate the edge probabilities, this graph serves as a gold standard. The other three graphs were generated using the functional similarity metrics to be compared. To make the PIN-like network more realistic some labels were hidden, as we don’t always know the exact function of every protein when we evaluate PINs.

Now that we have the results, we just need to see how all the coloured graphs compare to the black ones, or in our analogy: how all the kid’s drawings compare to what our house looks like. But because we are doing science, one drawing competition sadly won’t do. What if one child draws our house very well, but happens to do a bit worse exactly in the parts where the house is similar to a farm. We’d think he’s the guy to listen to, and start thinking a farm looks like a shed?!

What we really need, is more drawing competitions. Lots of them. And this is where I am happy I’m at a computer where running one of these competitions takes maybe a minute. To get a bit more confidence in the results, I ran every simulation 100 times, for 27 different sets of parameters. And the results? Well, that’s the best bit. The results say Tom was drawing a great farm all along. Tom, the kid you’ve seen grow up and been bringing to daycare for years now, he was right. You weren’t lying at all when you said it was a great farm. Only in real life he’s not called Tom, he’s called the Pandey method ;).

Antibodies are an invaluable class of therapeutic molecules — they have the capacity to bind any molecule (Martin and Thornton, 1996), and this comes from an antibody’s remarkable diversity (Georgiou et al., 2014). In particular, antibodies can bind their targets with high affinity, and most drug design campaigns seek to ‘mature’ an antibody, i.e. increase the antibody’s affinity for its target. However, the process of antibody maturation is, in experimental terms, time-consuming and expensive — if we had 6 CDRs (as in a typical antibody), with 10 residues each, and if you can have any of the 20 amino acids in the CDR positions, there are 20^60 mutants to test (and this is before considering any double or triple mutations!)

So hold on, what is affinity exactly? Affinity represents the strength of binding, and it’s calculated as either a ratio of concentrations, or as a ratio of rate constants, i.e.In the simplest affinity maturation protocol, three steps are compulsory:

1. Mutate the antibody’s structure correctly
2. Assess the impact of mutation on KD
3. Select and repeat.

For the past year, we have centralised around part 2 — affinity prediction. This is a fundamental aspect of the affinity maturation pipeline in order to rationalise ‘good’ and ‘bad’ mutations in the context of maturing an antibody. We developed a statistical potential, CAPTAIN; essentially the idea is to gather contact statistics that are represented in antibody-antigen complexes, and use this information to predict affinities.

But why use contact information? Does it provide anything useful? Based on analyses of the interfaces of antibody-antigen complexes in comparison to general protein-protein interfaces, we definitely see that antibodies rely on a different binding mode compared to general protein-protein complexes, and other studies have confirmed this notion (Ramaraj et al., 2012; Kunik and Ofran, 2013; Krawczyk et al., 2013).

For our methodology, we trained on general protein-protein complexes (as most scoring functions do!) and specifically on antibody-protein complexes from the PDB. For our test set of antibody-protein complexes, we outperformed 16 other published methods, though for our antibody-peptide test set, we were one of the worst performers. We found that other published methods predict antibody-protein affinities poorly, though they make better predictions for antibody-peptide binding affinities. Ultimately, we achieve stronger performance as we use a more appropriate training set (antibody-antigen complexes) for the problem in hand (predicting antibody-antigen affinities). Our correlations were by no means ideal, and we believe that there are other aspects of antibody structures that must be used for improving affinity prediction, such as conformational entropy (Haidar et al., 2013) and VH-VL domain orientation (Dunbar et al., 2013; Fera et al., 2014).

What’s clear though, is that using the right knowledge base is key to improving predictions for solving greater problems like affinity maturation. At present, most scoring functions are trained on general proteins, but this ‘one-fits-all’ approach has been subject to criticism (Ross et al., 2013). Our work supports the idea that scoring functions should be tailored specifically for the problem in hand.

 

Motivation

Recently I published a paper on OOMMPPAA, my 3D matched-molecular pair tool to aid drug discovery. The tool is available to try here, download here and read about here. OOMMPPAA aims to tackle the big-data problem in drug discovery – where X-ray structures and activity data have become increasingly abundant. Below I will explain what a 3D MMP is.

What are MMPs?

MMPs are two compounds that are identical apart from one structural change, as shown in the example below. Across a set of a thousand compounds, tens of thousands of MMPs can be found. Each pair can be represented as a transformation. Below would be Br >> OH. Within the dataset possibly hundreds of Br >> OH will exist.

 

 

 

 

 

 

Each of these transformations will also be associated with a change in a measurable compound property, (solubility, acidity, binding energy, number of bromine atoms…). Each general transformation (e.g. Br>>OH) therefore would have a distribution of values for a given property. From this distribution we can infer the likely effect of making this change on an unmeasured compound. From all the distributions (for all the transformations) we can then predict the most likely transformation to improve the property we are interested in.

The issue with MMPs

Until recently the MMP approach had been of limited use for predicting compound binding energies. This is for two core reasons.

1) Most distributions would be pretty well normally distributed around zero. Binding is complex and the same change can have both positive and negative effects.

2) Those distributions that are overwhelmingly positive, by definition, produce increased binding against many proteins. A key aim of drug discovery is to produce selective compounds that increase binding energy only for the protein you are interested in. So increasing binding energy like that is not overall very useful.

3D MMPs to the rescue

3D MMPs aim to resolve these issues and allow MMP analysis to be applied to binding energy predictions. 3D MMPs use structural data to place the transformations in the context of the protein. One method, VAMMPIRE, asks what is the overall effect of Br>>OH when it is near to a Leucine, Tyrosine and Tryptophan (for example). In this way selective changes can be found.

Another method by BMS aggregates these changes across a target class, in their cases kinases. From this they can ask questions like, “Is it overall beneficial to add a cyclo-proply amide in this region of the kinase binding site”.

What does my tool do differently?

OOMMPPAA differs from the above in two core ways. First, OOMMPPAA uses a pharmacophore abstraction to analyse changes between molecules. This is an effective way of increasing the number of observations for each transition. Secondly OOMMPPAA does not aggregate activity changes into general trends but considers positive and negative activity changes separately. We show in the paper that this allows for more nuanced analysis of confounding factors in the available data.

At this week’s group meeting I presented my recently published paper:

Knapp B, Dunbar J, Deane CM. Large scale characterization of the LC13 TCR and HLA-B8 structural landscape in reaction to 172 altered peptide ligands: a molecular dynamics simulation study. PLoS Comput Biol. 2014 Aug 7;10(8):e1003748. doi: 10.1371/journal.pcbi.1003748. 2014.

This paper was presented on the front page of PLoS Computational Biology:

The paper describes Molecular Dynamics simulations (MD) of T-cell receptor (TCR) / peptide / MHC complexes.

MD simulation calculate the movement of atoms of a given system over time. The zoomed-in movements of the TCR regions (CDRs) recognizing a peptide/MHC are shown here:

Often scientists perform MD simulations of two highly similar complexes with different immunological outcome. An example is the Epstein-Barr Virus peptide FLRGRAYGL which leads to induction of an immune response when presented by HLA-B*08:01 to the LC 13 TCR. If position 7 of the peptide is mutated to A then the peptide does not induce an immune reaction any-more.
In both simulations the TCR and MHC are identical only the peptide differs in one amino acid. Once the simulations of those two complexes are done one could for example investigate the root mean square fluctuation of both peptides to investigate if one of them is more flexible. And indeed this is the case:

Another type of analysis would be the solvent accessible surface area of the peptide or, as shown below, the CDR regions of the TCR beta chain:

… and again it is obvious that those two simulations strongly differ from each other.

Is it then a good idea to publish an article about these findings and state something like “Our results indicate that peptide flexibility and CDR solvent accessible surface area play an important in the activation of a T cell” ?

As we know from our statistics courses a 1 vs 1 comparison is an anecdotic example but those often do not hold true for larger sample sizes.

So, let’s try it with many more peptides and simulations (performing these simulations took almost 6 months on the Oxford Advanced Research Computing facility). We split the simulations in two categories, those which are more immunogenic and those which are less immunogenic:

Let’s see what happens to the difference in RMSF that we found before:

So much for “More immunogenic peptides are more/less flexible as less immunogenic ones” …

How about the SASA that we “found” before:

… again almost perfectly identical for larger sample sizes.

And yes, we have found some minor differences between more and less immunogenic peptides that hold true for larger sample sizes but non of them was striking enough to predict peptide immunogenicity based on MD simulations.

What one really learns from this study is that you should not compare 1 MD simulation against 1 other MD simulation as this will almost certainly lead to false positive findings. Exactly the same applies for experimental data such as x-ray structures because this is a statistical problem rather than a prediction based on. If you want to make sustainable claims about something that you found then you will need a large sample size and a proper statistical sample size estimation and power analysis (as done in clinical studies) before you run any simulations. Comparing structures is always massive multiple testing and therefore high sample sizes are needed to draw valid conclusions.

link pdf

The research I do revolves around how to break down protein interaction networks (PINs) into functional modules, or communities, using community detection methods. These communities are groups of proteins which interact more with one another than with the rest of the network. We decompose the PINs in this manner, as it is very difficult to determine how a protein functions through its interactions by simply looking at the entire network. The sheer amount of data in one of these networks, clouds the information we would actually like to see, as explained in my previous blog post.

In a journal club in August, I presented the overlapping community detection method Link Clustering by Ahn et al. which is the first community detection method I am aware of that assigns nodes to communities by clustering the edges between the nodes. I contrasted Link Clustering with the Affiliation Graph Model method proposed by Yang and Leskovec, as the authors use the same comparison technique to validate their methods. In a world where studies usually only work on a single network using a specific method, having two independent papers that use the same validation method is something that gets me much more excited than it probably should.

 

Link Clustering

Instead of assigning nodes to communities, Link Clustering focusses on the edges in a network. Clustering edges is essentially an equivalent problem to clustering nodes. This becomes obvious if you take a graph, decide to call all the edges “blobs” and say two of your new “blobs” have an edge between them if they share a node in the original network. This is called creating the “line graph” of the network and performing “blob” clustering on this is the same as performing edge clustering on the initial network.

To come back from my tangent, if an edge is assigned to community A, this means the nodes on either side of this edge are assigned community A by association. By assigning each edge to an individual community, Link Clustering creates overlapping node communities as shown in Figure 1 below.

In Link Clustering, edges are placed into the same community, based on similarity scores computed between all connected edges. Edge clusters are computed from these similarity scores using single-linkage clustering, where the edges with the highest similarity scores are iteratively merged into the same community. Using this method all edges end up in the same community in the end, so a threshold for the similarity score needs to be found at which the network is partitioned into communities in the best way. This threshold value represents a type of resolution parameter of the for community detection. At a low threshold there are few, large communities as many edges have been clustered together, while at a high threshold only highly similar edges are in the same community and there are many, small communities. Ahn et al. propose maximizing a function called the partition density to find the optimal resolution threshold. This function is simply a weighted average of the density of the communities. For those incredibly keen, it is given by the equation:

Here, represents the total number of edges in the network and and represent the number of edges and nodes in the community respectively.

All of this partitioning is based on a similarity score between two edges. The crux of this method, the scoring measure, is explained in Figure 2.

Affiliation Graph Model

The method which I would like to compare Link Clustering to is the Affiliation Graph Model (AGM). Briefly, this is a statistical community detection algorithm which generates overlapping communities. Node affiliations are recorded in the Community Affiliation Matrix shown in Figure 2.

The AGM has a probability of interaction for each community, denoted for community . For nodes and , which may share several community affiliations, the probability of interaction, is given by the equation:

where denotes a community in the set of communities shared by nodes and , . This model is be fitted to the network using a Metropolis-Hastings algorithm with a Markov chain constructed by pre-defined defined steps in the space of possible Community Affiliation Matrices (c.f. Yang and Leskovec 2012).

Result Comparison Method

Link Clustering itself has some very interesting results, especially relating to looking at real-world networks at different resolutions (which can be found in the paper). However, I want to focus on the validation method used in the paper which compares results generated from Link Clustering with that of other methods. This comparison method evaluates four different aspects of the partitions generated by different methods. These four aspects are: Community Quality, Overlap Quality, Community Coverage, and Overlap Coverage. The “Quality” aspects relate to network metadata indicating how good the obtained communities are, and the “Coverage” aspects relate to the amount of information extracted from a network.

The metadata referred to above is a general term for additional data available about the nodes in a network. In the case of PINs this metadata is related to the function of proteins in the network (their Gene Ontology annotations). Loosely, proteins with similar functions should be placed in the same community to improve the Community Quality measure. Overlap Quality concerns the boundaries between generated communities. If the functions assigned to proteins show similar boundaries between groups of functions, the Overlap Quality is high.

The “Coverage” values are more basic calculations. A partition has a high Community Coverage if the fraction of nodes assigned to communities with three or more nodes is high (non-trivial communities). A community of size two is uninformative, as it is the result of a single edge being in a community by itself. Overlap Coverage is simply the number of non-trivial community memberships per node. The two “Coverage” values are thus equal for non-overlapping community detection methods.

When comparing community detection methods, these four measures are computed for a partition generated by each method, and then rescaled so that the maximum score achieved by any method is 1 for each measure independently. These values are then added to give a score between 0 and 4 for each community detection method.

 

Results

The results shown in Figure 4 were generated by Yang and Leskovec to show their AGM outperforms Link Clustering (and other methods) on a variety of networks. While it is noteable that they used the same metric and networks used by Ahn et al. when proposing Link Clustering, this Figure must be taken with a pinch of salt. Yang and Leskovec acknowledge that the metric proposed by Ahn et al. rewards methods which find more communities in a network and thus do not fit the number of communities judged to be best by the AGM, but instead fit the same number as fitted in Link Clustering. Furthermore, it is peculiar that half of the networks used for comparison by Ahn et al. have disappeared in this comparison.

To conclude we can say that while it is very interesting that two papers use the same method to compare their methods to others for validation purposes, it wasn’t done completely accurately here. The comparison metric is however an interesting development to create a gold standard for method comparison. For my purposes, Community Quality is the most important, so maybe a weighted version of this may be more interesting.

 

The European Conference on Computational Biology was held in Strasbourg, France this year. The conference was attended by several members of OPIG including Charlotte Deane, Waqar Ali, Eleanor Law, Jaroslaw Nowak and Cristian Regep. Waqar gave a talk on his paper proposing a new distance measure for network comparison (Netdis). There were many interesting talks and posters at the conference, and brief summaries of the ones we found most relevant are given below.

The impact of incomplete knowledge on the evaluation of protein function prediction: a structured output learning perspective.

Authors: Yuxiang Jiang, Wyatt T. Clark, Iddo Friedberg and Predrag Radivojac

Chosen by: Waqar Ali

The automated functional annotation of biological macromolecules is a problem of computational assignment of biological concepts or ontological terms to genes and gene products. A number of methods have been developed to computationally annotate genes using standardized nomenclature such as Gene Ontology (GO). One important concern is that experimental annotations of proteins are incomplete. This raises questions as to whether and to what degree currently available data can be reliably used to train computational models and estimate their performance accuracy.

In the paper, the authors studied the effect of incomplete experimental annotations on the reliability of performance evaluation in protein function prediction. Using the structured-output learning framework, they provide theoretical analyses and carry out simulations to characterize the effect of growing experimental annotations on the correctness and stability of performance estimates corresponding to different types of methods. They also analyzed real biological data by simulating the prediction, evaluation and subsequent re-evaluation (after additional experimental annotations become available) of GO term predictions. They find a complex interplay between the prediction algorithm, performance metric and underlying ontology. However, using the available experimental data and under realistic assumptions, their results also suggest that current large-scale evaluations are meaningful and surprisingly reliable. The choice of function prediction methods evaluated by the authors is not exhaustive however and it is quite possible that other methods might be much more sensitive to incomplete annotations.

Towards practical, high-capacity, low-maintenance information storage in synthesized DNA.

Authors: Nick Goldman, Paul Bertone, Siyuan Chen, Christophe Dessimoz, Emily M. LeProust, Botond Sipos & Ewan Birney

Chosen by: Jaroslaw Nowak

This was one of the keynote speaker talks. One of the authors, Ewan Birney discussed how viable is storing digital information in DNA code. The paper talked about storing 739 kilobytes of hard-disk storage in the genetic code. The data stored included all 154 of Shakespeare’s sonnets in ASCII text, a scientific paper in PDF format, a medium-resolution colour photograph of the European Bioinformatics Institute in JPEG format, a 26-s excerpt from Martin Luther King’s 1963 ‘I have a dream’ speech in MP3 format and a Huffman code used in their study to convert bytes to base-3 digits (ASCII text). The authors accomplished this by first converting the binary data into base-3 numbers using Huffman coding (which represents the most common piece of information using the least bits). The base-3 numbers where then converted into a genetic code in such a way that produced no homopolymers. The authors also proved that they can read the encoded information and reconstruct the data with 100% accuracy.

Using DNA for information storage could very soon become cost-effective for sub-50 years archiving. The current costs of the process are $12,400/MB for writing and $220/MB for reading information, with negligible costs of copying. Nevertheless, if the current trends persist, we could see a 100 – fold drop in costs in less than a decade. The main advantages of storing information in DNA is the low maintenance and durability of the medium (intact DNA fragments have been recovered from samples that are tens of thousands years old) as well as little physical space required to store the information (~2.2 PB/g)

 

PconsFold: Improved contact predictions improve protein models.

Authors: Michel M, Hayat S, Skwark MJ, Sander C, Marks DS and Elofsson A.

Chosen by: Eleanor Law

De novo structure prediction by fragment assembly is a very difficult task, but can be aided by contact prediction in cases where there is plenty of sequence data. Contact prediction has also significantly improved recently, using statistical methods to separate direct from indirect contact information.

PSICOV and plmDCA are two such methods, providing contacts which can be used by software such as Rosetta as an additional energy term. PconsFold combines 16 different sets of contact predictions by these programs, built from different sequence alignments, with secondary structure and solvent accessibility prediction. The output of the deep learning process on these inputs is more reliable that the individual contact predictions alone, and produces more accurate models. The authors found that using only 2,000 decoys rather than 20,000 did not greatly harm their results, which is encouraging as the decoy generation stage is the particularly resource intensive stage. Using a balance between the Rosetta energy function and weight of contact prediction, the optimal number of constraints to include was around 2 per residue, compared to 0.5 per residue for PSICOV or plmDCA alone.

The PconsFold pipeline is not always able to make full use of the contact prediction, as accuracy of contact prediction on the true structure can be higher than that in the model. This is a case where the conformational search is not effective enough to reach the correct answer, though it would be scored correctly if it were obtained. All-beta proteins are the most difficult to predict, but PconsFold compares favourably to EVfold-PLM for each of the mainly alpha, mainly beta, and alpha & beta classes.

In the group meeting on the 20th of August, I presented the paper by Vazquez et al (2002). This was one of the first papers proposing the duplication divergence model of evolution for protein interaction networks, and thus has had a significant impact on the field, inspiring many variants of the basic model. The paper starts out by noting that the yeast protein network has the ‘small world’ property – following along links in the network, it requires only a handful of steps to go from any one protein to any other. Another property is the manner in which the links are shared out among the various proteins: empirically, the probability that a protein interacts with k other proteins follows a power-law distribution.

Vazquez et al. show how evolution can produce scale-free networks. They explore a model for the evolution of protein networks that accurately reproduces the topological features seen in the yeast S. cerevisae. As the authors point out, proteins fall into families according to similarities in their amino-acid sequences and functions, and it is natural to suppose that such proteins have all evolved from a common ancestor. A favoured hypothesis views such evolution as taking place through a sequence of gene duplications – a relatively frequent occurrence during cell reproduction. Following each duplication, the two resulting genes are identical for the moment, and naturally lead to the production of identical proteins. But between duplications, random genetic mutations will lead to a slow divergence of the genes and their associated proteins. This repetitive, two-stage process can be captured in a relatively simple model for the growth of a protein interaction network .

Simulations by the author show that the model, starting out with a seed capture the degree distribution of the yeast network with high fidelity (Fig 1) and also possesses the quality of high tolerance to random node removal seen in biological networks. While the results are more qualitative in nature, the model still serves as the basis of most biologically rooted explanations of protein network evolution, with minor improvements. Some of these additions have been the use of asymmetric divergence, whole genome duplication events as well as interaction site modelling. As the jury is still out on what model (if any) best fits current interaction data, the basic model is still relevant as a benchmark for newer models.

The work of our molecular dynamic expert, Bernhard Knapp, has made the front page of the PLOS Computational Biology . The image shows how CDR3 loops scan a peptide/MHC complex (Figure below: Snapshot from the website). Immune cells in the human body screen other cells for possible infections. The binding of T-cell receptors (TCR) and parts of pathogens bound by major histocompatibility complexes (MHC) is one of the activation mechanisms of the immune system. There have been many hypotheses as to when such binding will activate the immune system. In this study we performed the, to our knowledge, largest set of Molecular Dynamics simulations of TCR-MHC complexes. We performed 172 simulations each of 100 ns in length. By performing a large number of simulations we obtain insight about which structural features are frequently present in immune system activating and non-activating TCR-MHC complexes. We show that many previously suggested structural features are unlikely to be causal for the activation of the human immune system.