Sampling Conformations of Antibodies using MOSAICS

Much work has been done to study the conformational changes taking place in antibodies, particularly during the event of binding to an antigen. This has been done through comparison of crystal structures, circular dichroism, and recently with high resolution single particle electron microscopy. The ability to resolve domains within an antibody from single particles without any averaging  made it possible to show distributions of properties such as the shape of a Fab domain, measured by the ratio of width to length. Some of the variation in structure seen involves very large scale motions, but it is not known how conformational changes may be transmitted from the antigen binding region to the Fc, and therefore influence effector function. Molecular dynamics simulations have been performed on some large antibody systems, however none have been possible on a time scale which would be able to provide information on the converged distributions of large scale properties such as the angle between the Fab and Fc fragments.

In my short project with Peter Minary, I used MOSAICS to investigate the dynamics of an antibody Fab fragment, using the coarse-grained natural move Monte Carlo approach described by Sam a few weeks ago. This makes it possible to split a structure into units which are believed to move in a correlated way, and propose moves for the components of each region together. The rate of sampling is accelerated in degrees of freedom which may have functional significance, for example the movement of the domains in a Fab fragment relative to one another (separate regions shown in the diagram below). I used ABangle to analyse the output of each sampling trajectory and observe any changes in the relative orientations of The VH and VL domains.

Region definitions for MOSAICS

Fab region definitions for MOSAICS

Of particular interest would be any correlations between conformational changes in the variable and constant parts of the Fab fragment, as these could be involved in transmitting conformational changes between remote parts of the antibody. We also hoped to see in our model some effect of including the antigen in the simulation, bound to the antibody fragment as seen in the crystal structure. In the time available for the project, we was able to  set up a model representing the Fab fragment and run some relatively short simulations to explore favoured conformational states and see how the set up of regions affects distributions seen. In order to draw conclusions about the meaning of the results, a much greater number of simulations will need to be run to ensure sampling of the whole conformational space.

Computational Antibody Affinity Maturation

In this week’s journal club, we reviewed a paper by Lippow et al. in Nature Biotechnology, which features a computational pipeline that is capable of maturing antibodies (Abs) by up to 140-fold. The paper itself discusses 4 test case Abs (D44.1, cetuximab, 4-4-20, bevacizumab) and uses changes in electrostatic energy to identify favourable mutations. Up to the point when this paper was published back in 2007, computational antibody design was an (almost) unexplored field of research – except for a study by Clark et al. in 2006, no one else had done anything like the work presented in this paper.

The idea behind the paper is to identify certain positions within the Ab structure for mutation and hopefully find an Ab with a higher binding affinity.

The idea behind the paper is to identify certain positions within the Ab structure for mutation and hopefully find an Ab with a higher binding affinity.


Briefly speaking, the group generated a mutant Ab-antigen (Ag) complex using a series of algorithms (dead-end elimination and A*), which was then scored by the group’s energy function for identifying favourable mutations. Lippow et al. used the electrostatics term of their binding affinity prediction in order to estimate the effects of mutations on an Ab’s binding affinity. In other words, instead of examining their entire scoring function, which includes terms such as van der Waal’s energy, the group only used changes in the electrostatic energy term as an indicator for proposing mutations. Overall, in 2 of the 4 mentioned test cases (D44.1 & cetuximab), the proposed mutations were experimentally tested to confirm their computational design pipeline – a brief overview of these two case studies will be described.


In the case of the D44.1 anti-lysozyme Ab, the group proposed 9 single mutations by their electrostatics-based calculation method; 6/9 single mutants were confirmed to be beneficial (i.e., the mutant had an increased binding affinity). The beneficial single mutants were combined, ultimately leading to a quadruple mutant structure with a 100-fold improvement in affinity. The quadruple mutant was then subjected to a second round of computer-guided affinity maturation, leading to a new variant with six mutations (effectively a 140-fold improvement over the wild-type Ab). This case study was a solid testimony to the validity of their method; since anti-lysozyme Abs are often used as model systems, these results demonstrated that their design pipeline had taken, in principle, a suitable approach to maturing Abs in silico.

The second case study with cetuximab was arguably the more interesting result. Like the D44.1 case above, mutations were proposed to increase the Ab’s binding affinity on the basis of the changes in electrostatics. Although the newly-designed triple mutant only showed a 10-fold improvement over its wild-type counterpart, the group showed that their protocols can work for therapeutically-relevant Abs. The cetuximab example was a perfect complement to the previous case study — it demonstrated the practical implications of the method, and how this pipeline could potentially be used to mature existing Abs within the clinic today.

Effectively, the group suggested that mutations that either introduce hydrophobicity or a net charge at the binding interface tend to increase an Ab’s binding affinity. These conclusions shouldn’t come with huge surprise, but it was remarkable that the group had reached these conclusions with just one term from their energy function.


Effectively, the paper set off a whole new series of possibilities and helped us to widen our horizons. The paper was by no means perfect, especially with respect to predicting the precise binding affinities of mutants – much of this error could be bottled down to the modelling stage of their pipeline. However, the paper showed that computational affinity maturation is not just a dream – in fact, the paper showed that it’s perfectly doable, and immediately applicable. Interestingly, Lippow et al.’s manipulation of an Ab’s electrostatics seemed to be a valid approach, with recent publications on Ab maturation showing that introducing charged residues can enhance binding affinity (e.g. Kiyoshi et al., 2014).

More importantly, the paper was a beautiful showcase of how computational analyses could inform the decision making process in an in vitro framework, and I believe it exemplified how we should approach our problems in bioinformatics. We should not think of proteins as mere text files and numbers, but realise that they are living systems, and we’re not yet at a point where we fully understand how proteins behave. This shouldn’t discourage us from research; instead, it should give us the incentive to take things more slowly, and develop a method/product that could be used to solve greater, pragmatic problems.

Le Tour de Farce v2.0

In what is becoming the highlight of the year and a regular occurrence for the OPIGlets, Le Tour de Farce – The annual OPIG bike ride, took place on the 4th of June. Now in its 2.0 revision but maintaining a route similar to last year, 9.5 miles and several pints later, approximately 20 of us took in some distinctly pretty Oxfordshire scenery, not to mention The White Hart, The Trout, Jacobs Inn and for some, The One and The Punter too.




Antibody modeling via AMA II and RosettaAntibody


Protein modeling is one of the most challenging problems in bioinformatics. We still lack a clear theoretical framework which would allow us to link linear protein sequence to its native 3D coordinates. Given that we only have the structures for about a promile of the known seqs, homology modeling is still one of the most successful methods to obtain a structure from a sequence. Currently, using homology modeling and the 1393 known folds we can produce models for more than half known domains. In many cases this is good enough to get an overall idea of the fold but for actual therapeutic applications, there is still a need for high-resolution modeling.

There is one group of molecules whose properties can be readily exploited via computational approaches for therapeutic applications: antibodies.  With blockbuster drugs such as Humira, Avastin or Remicade, they are the leading class of biopharmaceuticals. Antibodies share a great degree of similarity with one another (<50-60% sequence identity) and there are at least 1865 antibody structures in the PDB. Therefore, homology modeling of these structures at high resolution becomes tractable, as exemplified by WAM and PIGS. Here, we will review the antibody modeling paradigm using one of the most successful antibody modeling tools, RosettaAntibody, concluding with the most recent progress from AMA II (antibody CASP).

General Antibody-antigen modeling

Modeling of antibody structures can be divided into the following steps:

  1. Identification of the Framework template
  2. Optimizing Vh/Vl orientation of the template
  3. Modeling of the non-H3 CDRs
  4. Modeling of H3

Most of the diversity of antibodies can be found in the CDRs. Therefore, the bulk of the protein can be readily copied from the framework region. This however needs to undergo an optimization of the Vh/Vl orientation. Prediction of the CDRs is more complicated since they are much more variable than the rest of the protein. Non-H3 CDRs can be modeled using canonical structure paradigms. Prediction of H3 is much more difficult since it does not appear to follow the canonical rules.

When the entire structure is assembled, it is recommended to perform refinement using some sort of relaxation of the structure, coupled with an energy function which should guide it.


RosettaAntibody protocol roughly follows this described above. In the first instance, an appropriate template is identified by highest BLAST bit scores. The best heavy and light chains aligned to the best-BLAST-scoring Fv region. The knowledge-base here is a set of 569 antibody structures form SACS with resolutions 3.5A and better. The Vh/Vl orientation is subsequently refined using local relaxation, guided by Charmm.

Non-H3 CDRs are modeled using the highest-scoring BLAST hit of the same length. Canonical information is not taken into account. Loops are grafted on the framework using the residues overlapping with the anchors.

H3 loops are modeled using a fragment based approach. The fragment library is Rosetta+H3 from the knowledge base of antibody structures created for the purpose of this study. The low-resolution search consists of Monte Carlo attempts to fit 3-residue fragments followed by Cyclic Coordinate Descent loop closure. This is followed by high resolution search when the H3 loop and Vh/Vl are repacked using a variety of moves.

Each decoy coming from the repacking is scored using Rosetta function. The lower the Rosetta score the better the decoy (according to Rosetta).


RosettaAntibody can produce high-quality models (1.4A) on its 54 structure benchmark test. The major limitation of the method (just like any other antibody modeling method) is the H3 loop modeling. It is believed that H3 is the most important loop and therefore getting this loop right is a major challenge.

Right framework and the correct orientation of Vh/Vl have a great effect on the quality of H3 predictions. When the H3 was modeled on using the correct framework, the predictions are order of magnitude better than by using the homology model. This was demonstrated using the native recovery in RosettaAntibody study as well as during ‘Step II’ of the Antibody Modeling assessment where participants were asked to model H3 using the correct framework.

Journal club (Bernhard Knapp): MMPBSA Binding Free Energy Calculations

This week’s topic of the Journalclub was about Molecular Mechanics Poisson−Boltzmann Surface Area (MMPBSA) binding free energy calculations between ligand and receptor using Molecular Dynamics simultions (MD). As an example I selected:

David W. Wright, Benjamin A. Hall, Owain A. Kenway, Shantenu Jha, and Peter V. Coveney. Computing Clinically Relevant Binding Free Energies of HIV-1 Protease Inhibitors. J Chem Theory Comput. Mar 11, 2014; 10(3): 1228–1241

The first question is: Why do we need such rather complex and computationally expensive approaches if other (e.g. empirical) scoring functions can do similar things? The main challenges thereby is that simple scoring functions often do not work very well for systems where they were not calibrated on (e.g. Knapp et al. 2009 ( The reasons for that are manifold. MD-based approaches can improve two major limitations of classical docking/scoring functions:

1) Proteins are not static. Ligand as well as receptor can undergo various slightly different configurations even for one binding site. Therefore the view of scoring one ligand configuration against one receptor configuration is not the whole picture. The first improvement is to consider a lot of different configurations for one position score of the ligand:


2) A more physics based scoring function can be more reliable than a simple and run-time efficient scoring function. On the basis of the MD simulations a variety of different terms can be deduced. These include:


- MM stands for Molecular Mechanics. It’s internal energy includes bond stretch, bend, and torsion. The electrostatic part is calculated using a Coulomb potential while the Van der Waals term is calculated using a Lennard-Jones potential.
- PB stands for Poisson−Boltzmann. It covers the polar solvation part i.e. the electrostatic free energy of solvation.
- SA stands for Surface Area. It covers the non-polar solvation part via a surface tension weighted solvent accessible surface area calculation.
- TS stands for the entropy loss of the system. This term is necessary because the non-polar solvation incorporates an estimate of the entropy changes implicitly but does not account for an entropy change upon receptor/ligand formation in vacuo. This term is calculated on the basis of a normal mode analysis.

If all these terms are calculated for each single frame of the MD simulations and those single values are averaged an estimate of the binding free energy of the complex can be obtained. However, this estimate might not represent the actual mean of the spatial distribution. Therefore at least 50 replica MD simulations are needed per investigated complex. In this aspect replica means an identically parameterized simultion of the same complex where only the inital forces are assinged randomly.

On the basis of the described MMPBSA-TS approach in combination with 50 replicas the authors achieve a reasonable correlation (0.63) for the 9 FDA-approved HIV-1
protease inhibitors with know experimental binding affinities. If the two largest complexes are excluded the correlation improves to an excellent value (0.93).

In a current study we are using the same methodology for peptide/MHC interactions. This system is completely different from the protease inhibitor study of Wright et al.: The ligands are peptides and the binding site is a groove consisting of two alpha-helices. The methods was applied as it is (without calibration or any kind of training). Prelimiary data still shows a high correlation with experimental values for the peptide/MHC system. This indicates that this MMPBSA approach can yield reliable predictions for very different systems without further modification.

Natural Move Monte Carlo: Sampling Collective Motions in Proteins

Protein and RNA structures are built up in a hierarchical fashion: from linear chains and random coils (primary) to local substructures (secondary) that make up a subunit’s 3D geometry (tertiary) which in turn can interact with additional subunits to form homomeric or heteromeric multimers (quaternary). The metastable nature of the folded polymer enables it to carry out its function repeatedly while avoiding aggregation and degradation. These functions often rely on structural motions that involve multiple scales of conformational changes by moving residues, secondary structure elements, protein domains or even whole subunits collectively around a small set of degrees of freedom.

The modular architecture of antibodies, makes them amenable to act as an example for this phenomenon. Using MD simulations and fluorescence anisotropy experiments Kortkhonjia et al. observed that Ig domain motions in their antibody of interest were shown to correlate on two levels: 1) with laterally neighbouring Ig domains (i.e. VH with VL and CH1 with CL) and 2) with their respective Fab and Fc regions.

Correlated Motion

Correlated motion between all residue pairs of an antibody during an MD simulation. The axes identify the residues whereas the colours light up as the correlation in motion increases. The individual Ig domains as well as the two Fabs and the Fc can be easily identified. ref: Kortkhonjia, et al., MAbs. Vol. 5. No. 2. Landes Bioscience, 2013.

This begs the question: Can we exploit these molecular properties to reduce dimensionality and overcome energy barriers when sampling the functional motions of metastable proteins?

In 2012 Sim et al. have published an approach that allows for the incorporation of these collective motions (they call them “Natural Moves”) into simulation. Using simple RNA model structures they have shown that explicitly sampling large structural moves can significantly accelerate the sampling process in their Monte Carlo simulation. By gradually introducing DOFs that propagate increasingly large substructures of the molecule they managed to reduce the convergence time by several orders of magnitude. This can be ascribed to the resulting reduction of the search space that narrows down the sampling window. Instead of sampling all possible conformations that a given polynucleotide chain may take, structural states that differ from the native state predominantly in tertiary structure are explored.

Reduced Dimensionality

Reducing the conformational search space by introducing Natural Moves. A) Ω1 (residue-level flexibility) represents the cube, Ω2 (collective motions of helices) spans the plane and Ω3 (collective motions of Ω2 bodies) is shown as a line. B) By integrating multiple layers of Natural Moves the dimensionality is reduced. ref: Sim et al. (2012). PNAS 109(8), 2890–5. doi:10.1073/pnas.1119918109

It is important to stress, however, that in addition to these rigid body moves local flexibility is maintained by preserving residue level flexibility. Consequently, the authors argue, high energy barriers resulting from large structural rearrangements are reduced and the resulting energy landscape is smoothened. Therefore, entrapment in local energy minima becomes less likely and the acceptance rate of the Monte Carlo simulation is improved.

Although benchmarking of this method has mostly relied on case studies involving model RNA structures with near perfect symmetry, this method has a natural link to near-native protein structure sampling. Similarly to RNA, proteins can be decomposed into local substructures that may be responsible for the main functional motions in a given protein. However, due to the complexity of protein motion and limited experimental data we have a limited understanding of protein dynamics. This makes it a challenging task to identify suitable decompositions. As more dynamic data emerges from biophysical methods such as NMR spectroscopy and databases such as are extended we will be able to better approximate protein motions with Natural Moves.

In conclusion, when applied to suitable systems and when used with care, there is an opportunity to breathe life into the static macromolecules of the pdb, which may help to improve our understanding of the heterogeneous structural landscape and the functional motions of metastable proteins and nanomachines.

Protein Folding: Man vs Machine

In 1996 Gary Kasparov, the reigning world chess champion, played IBM’s Deep blue, a computer whose sole purpose was to play chess better than any human. Losing the first match, Gary sprung back swiftly defeating Deep Blue 4-2 over the remaining matches. However, his success was short lived. In a rematch with an updated Deep Blue the following year, the score was 3.5-2.5 to the computer. The media (and IBM) declared this as a pivotal moment in history, where a machine had proven itself better than humanities champion at a game deemed a highly intellectual pursuit. The outcry was that the age of machines had arrived. Was it true? Should humanity have surrendered to machine overloads at that moment? Obviously the answer is a large and resounding no. However, this competition allows for insightful comparison between the manner in which humans and computers play chess and think. By comparing the two, we learn the strengths and weaknesses of both parties from which we can make combined approaches that may exceed either.

Firstly, lets discuss the manner in which a computer “plays” chess. They simply search all possible configurations of moves that are available and pick the most optimal. However, things are not that simple. Consider only the opening sequence, there are 20 possible moves a player can make, so after only a single move by each player there is 400 possible chess positions. This count grows exponentially fast, after 5 moves by each player there is approximately 5 million combinations. For example, it was estimated that Deep Blue could analyse 2 million positions per second. However, since this is not nearly fast enough to examine all possible games from start to end in a reasonable time scale, computers cannot foresee lines of plays which are far in the distance. To overcome this, in the early game the computer will use a reference table developed by grandmasters that list both common openings and the assumed best manner to respond to them. Obviously, these are only assumed as optimal and have never been completely tested. In short, machines participate through a brute force, utilising their intricate ability to perform calculations at high speed to find the best move. However, the search is too large in the initial and end stages of a game to be completely thorough, a reference table is instead used to “inform” of the correct move at these times.


While a human can quite easily see that the following board leads to a draw, computers cannot draw the same conclusion without huge effort.

In contrast, human players use far more visual and spatial recognition alongside both memory and calculation to pick their moves. Like a computer, a player will analyse a portion of the moves available at any given moment. Though since a human cannot compare on computation speed to that of a computer, they cannot analyse nearly the same magnitude of moves. Hence, this subset of moves chosen for analysis must contain the most optimal move(s) to compete against the computer’s raw power. This is where the visual and spatial recognition abilities of humans come to bare. Firstly, a human can easily dissect the board into pieces worth considering and those to be ignored. For example, consider a possible move that would result in your queen being exposed and then taken. A human would conclude this as bad (normally) and discard further moves leading from such a play. A computer, however, would explore the resultant board state. One can see how this immediately and drastically reduces the required search. Another human ability is that a player will often be able to able to see sub-structures within a full set-up that are common in the game and hence can be processed in a known manner. In other words, the game is broken down into fragments which can be processed far easier and with less computation. Obviously, both of the above techniques rely on prior knowledge of chess to be useful, but they based upon our human ability to perceive both the substructure of the game and the overall picture with relative ease.

So how does all this chess talk relate to protein folding? In 2010, the Baker group and creators of the ROSETTA protein fold prediction program produced the protein folding game “Foldit”. In Foldit the general public could attempt to fold proteins for themselves and try to get closer to the native structure than the computer algorithms. Obviously, simplified in presentation to that of academic structural biology, it was hoped that the visual and spatial reasoning abilities of humans, the same ones that differentiated them from machines at chess, would prove useful in protein structure prediction. A key issue within ROSETTA drove this train of thought, the fact that is is relatively bad at exploring fully the confirmation space. Often, it will get stuck in the one general configuration and not explore the fold space fully. Furthermore, due to the size of configuration space, this is not easily overcome with simulated annealing due to the sheer scale of the problem. The ability of humans to view the overall picture meant that it should be easier for them to see other possible configurations. As end goals for Foldit, it was hoped that structures that proved unsolvable by current algorithms would be solved by humans and also that new techniques would emerge as “moves” employed by players to achieve high scores could be studied.

To make a comparison of the structures produced by Foldit players and ROSETTA viable, the underlying energy “scores” that judge a structure is the same between the programs. It is assumed, though is not always true, that the better the score the closer you are to the native fold. In addition,  Foldit players were also able to use a set of optimisation tools that were deterministic and would alter the backbone and side chains to the most optimal local configuration to the arrangement the player would make. This meant that players could focus predominantly on altering the overall structure of the protein rather than the fine detail, such as the position of sidec hains. To make the game as approachable as possible, technical terms were replaced by common analogues and visual cues where displayed to highlight poor scoring areas of the protein. For example, clashes between atoms are shown via large spiked red orbs, while the backbone is coloured from green to red depending on how well buried the hydrophobic residues on that segment are. To drive players, gamification elements were also included such as leader boards and rewarding “fireworks” as graphical effects.

To objectively compare the ability of the player base to that of the ROSETTA algorithm, they performed blind predictions on a set of 10 proteins whose structure were not in the public domain. This was run in a similar manner to CASP for those familiar with that set-up. The results exemplified the innate human ability of visual and spatial recognition. In 5 of the cases the playerbase performed significantly better than the ROSETTA program. In 3 of the cases they performed similar. And in the remaining 2 cases the ROSETTA algorithm performed better, though in both of these the model produced was still extremely far from the native structure. Looking through the cases individually, it was identified that the most crucial element used by players was that they were able to deal with large rearrangements that ROSETTA struggled to deal with, including register shifts and strand swapping. This highlights the ability of humans to view the overall picture and to persevere through “bad scoring patches” to reach a more optimal configuration.


Comparison of foldit player’s solutions (green) to ROSETTA’s solutions (red) and the native 2KPO protein structure (blue). The players correctly identified a strand swap needed to reach the native form, while this large reconfiguration was not seen by ROSETTA.

Since the release of the game and the accompanying paper in 2010, Foldit has received much praise in conveying the field of protein folding in an approachable manner to so many people. In addition, the player base has contributed to science as whole. In 2011 the player base successfully solved the structure of a M-PMV protein, a retrovirus whose structure was unobtainable via normal means. Then in 2012, by analysing the common set of moves employed by the player base, they collectively produced an algorithm that outperforms previously published fold prediction methods. Personally, I think of Foldit as a fun and relative intuitive game that introduces the core elements of the protein folding problem. As to its scientific merit, I’m unsure as to how much impact it will continue to have. As Saulo discussed last week, if infinite monkeys have infinite time then Shakespeare will be reproduced. Likewise, if enough people manipulate a protein structure, eventually the best structure will be found. Though who am I to judge, if people find the game fun, then there are far worse past-times one can have than trying to solve structures. As a finishing note I would be extremely interested in using Foldit to teach structural biology in the future, though feel it is overall too simple for a university setting.

de novo Protein Structure Prediction software: an elegant “monkey with a typewriter”

In this week’s OPIG group meeting, I discussed the inner-works and the algorithm behind ROSETTA, one of the most well-known software for de novo protein structure prediction.

Before we even attempt to understand how ROSETTA works, let us start with a theorem.

Theorem: given an infinite number of monkeys with typewriters and an infinite amount of time, they are very likely to recreate the works of William Shakespeare.

Monkey with a typewriter… Time to write that Shakespeare!

Well, let us be a little more modest and attempt to recreate just a phrase of old Bill, instead of his whole works:

“The fool doth think he is wise, but the wise man knows himself to be a fool.”

Well, if we exclude spaces and punctuation marks, that leaves us 58 positions in our phrase (the length of the quote). Considering we have 26 possible letters for each position, we would expect to generate this phrase at random once every of 26^58 times. Wow!

That means that we need to evolve from monkeys (pun intended) and appeal to our over-developed encephalon!

In order to steer our Monkey typewriter, we can reduce this problem to a Global Optimisation problem. In a Global Optimisation problem, we define a function f (named an objective function) which we want to minimise for a given set of parameters x. Bare in mind that if we want to maximise a given function fwe can define g = -f 

In a global optimisation problem, we are interested in finding the values of X that minimise the function f(X).

Now, all we need is to define an objective function in order to guide our Monkey typewriter towards the right answer.

Let us define the following objective function: given our Shakespearean phrase and a sequence of 58 letters, the value of the objective function equals the number of letters that are different between the phrase and the sequence of letters.

We can now proceed to define a slightly more refined Monkey Typewriter:

1- Start with a random sequence of letters.
2- WHILE sequence != shakespearean_phrase:
3-________ Select a random position in the sequence.
4-________ Assign a new letter to that position.
5-________ IF score of new sequence < score of old sequence:
6-__________________ Accept the change.
7-________ ELSE:
8-__________________ Discard the change.

This way we can steer our Monkeys and reduce the time it would take to generate our Shakespearean phrase to a more feasible time.

Now, let’s talk about protein structure prediction (PSP). More specifically, let us talk about de novo protein structure prediction (different flavours of protein structure prediction have been discussed previously here).

One of the great ideas behind the creators of ROSETTA, was to use a combination of two different techniques to address the big problems of protein structure prediction:

1- Problem number #1 of PSP is the size of the conformational space. A protein can be represented by it’s backbone atoms, which, in turn, can be reconstructed from a sequence of torsion angles. A set of 3 torsion angles can be used to represent every protein residue. Therefore, for a protein with 100 residues, we would have a total of 300 angles. If we approximate each angle to assume one of 360 values (degrees), that gives us 360^300 possible conformations (not huge at all, han?).

One of the main ideas behind ROSETTA was to reduce the search space by using fragments extracted from known structures. The use of fragments restricts the possible angles to a set of values that are known to occur in nature. Therefore, instead of looking at 360^300 possible angles, we deal with a much more feasible search space.

The name ROSETTA is based on the Rosetta Stone, an archaeological artefact that allowed modern civilisation to interpret and convert between different alphabets. In reality, ROSETTA can be seen as a very elegant monkey typewriter. ROSETTA uses sequence and structure similarity to define a structural alphabet. For every single position in our protein sequence, we have a set of fragments extracted from now protein structures to represent that position.  Originally, each position would be represented by 25 fragments (letters?). If you combine the different pieces of known structures in the right order, you will get your Shakespearean Phrase in the end (the correct Protein Structure!).

2- Well, we still have a pretty big conformational space considering we have 25 fragments per position (approximately 25^100 possible conformations, for a protein with 100 residues). The second technique employed by ROSETTA is Simulated Annealing.

Simulated Annealing is a Global Optimisation heuristic. It attempts to find a good enough solution to the problem of minimising a given function f. It is very similar to our Monkey Typewriter algorithm. The main difference is that Simulated Annealing implements some tricks to avoid local minima entrapment. In simpler terms, if we ONLY accept favourable changes (Line 5 of Monkey Typewriter pseudo-code), once we reach a local minimum, we get trapped. No possible change would lead to an improvement, yet we are still far from finding the global minimum.

In order to mitigate that entrapment effect, Simulated Annealing defines a probability of accepting an unfavourable change. This probability is higher at the beginning of the simulation and it becomes lower and lower as the simulation progresses. This process is usually referred to as “cooling down”.

Ok! So we reduced our PSP problem to an elegant Monkey Typewriter. We have our Monkeys working to create the best possible Shakespeare, in a pretty clever and sophisticated manner. Well, we should be able to create some fine piece of literature, correct?

Not quite!

There are still several problems with this whole pipeline. I will mention a few:

  • When you define your structural alphabet, you may not have the right fragment to represent a certain position. This would be the same as trying to get to a Shakespearean phrase without using vowels for the first 10 letters or only using consonants in the middle of the sentence. It would never happen…
  • Despite the many efforts to define a very good objective function, no current software presents a function that truly mimics the behaviour of an energy function. This implies that we have a vague idea of how the Shakespearean phrase should look like, but we cannot precisely pinpoint where each letter goes.
  • No matter how elegant our Monkey typewriter becomes, the combinatorial problem still persists. We are still dealing with 25^100 possible conformations and it is impossible to try every single conformation.
  • The objective function, if plotted in a graph, would look completely hideous (unlike the picture above). We are talking about a gigantic multi-dimensional surface, filled with local minima that confuse and entrap our simulations. Combine that with the fact that our objective function is not accurate and you waste most of your computing power into generating solutions that are completely useless.
  • Another common technique to address the previous limitations is to increase the number of Monkeys in order to speed up the search process. If you use thousands and thousands of Monkeys (multiple runs of ROSETTA), each individual Monkey will get to a local minimum (decoy = something that looks like a phrase). In recent years, tens of thousands of decoys are generated in order to predict a single structure. A new problem arises, because out of these tens of thousands of phrases, we cannot tell apart Hamlet from Twilight. We don’t know which Monkeys got close to the right answer. All we know is that for some cases some of them did.

In conclusion, de novo Protein Structure Prediction still has a long way to go.

MAMMOTH: a case study in protein structure alignment

I’ve talked about protein structure alignment before in the context of a rather novel, mathematical approach. This time I wanted to revisit the topic in a general sense, using a more established algorithm as a case study. MAMMOTH stands for Matching Molecular Models Obtained from Theory and was first published in 2002. Since then it has been cited nearly 400 times and the underlying algorithm has been extended to a multiple alignment program: MAMMOTH-mult.

Establishing biologically relevant and consistent alignments between protein structures is one of the major unsolved problems in computational bioinformatics. However, it’s an important part of many challenges that we face: such as establishing homology between distantly related proteins, functional inference for unannotated proteins, and evaluating the accuracy of models of predicted structure for competitions such as CASP.

Problem Outline

In essence the problem of protein structure alignment can be outlined by considering two ordered sets of coordinates, A = {a1,a2,…,an} and B = {b1,b2,…,bm}, representing points in 3D space. In most cases these points will be the location of the Cα atoms along each structure’s backbone. The sets A and B might be completely different lengths and, if an alignment exists, are almost certainly orientated differently compared to each other.


Establishing an alignment between these sets is equivalent to two steps:

  1. Establish a match M = {(ai,bj) | ai ∈ A, bj ∈ B}
  2. Rotate and translate A onto B so that equivalent atoms are as close as possible.

Of course, it is not immediately clear how to best fulfill these requirements. In particular, we don’t really know what features to prioritise for a biologically relevant match. Should we try to match secondary structure elements and what penalty should we attach to mismatched elements? How about maintaining the correct hydrogen bonding patterns between matched residues? And how much weight should we put on the matched atoms being consecutive in each set (i.e. how should we penalise gaps)?

The second step is equally ambiguous. Especially as there is no consensus on what the correct interpretation of close is. Minimising the RMSD between equivalent atoms is a popular choice of distance measure. However, as the MAMMOTH paper points out, RMSD is often dominated by the mismatched portions of remotely related structures and is thus largely inappropriate in these cases. Furthermore, even if we have a well-defined distance metric, should the superposition prioritise minimising the distances between nearly identical parts of the different structures, at the expense of less similar substructures? Or should the emphasis be on maintaining as lengthy a match as possible at the possible cost of a lower closeness of fit? How about the relative importance of a close fit for atoms in the core of the structure vs. those on the surface?

The majority of these questions remain unanswered and as a result it is often hard to validate alignments as we simply do not know what the right answer is. In fact, in many cases, manual analysis is preferred over any of the available computational techniques.

In this post I’ll go through how the MAMMOTH algorithm approaches each of these steps. For many of the above questions MAMMOTH does not postulate a solution, primarily because, as its name suggests, it was designed to assess prediction models which are often at low resolutions and lacking secondary structure or hydrogen bonding information. I think it’s important to keep these questions in mind, but there’s certainly no necessity to design a programme which deals with them all.

Step 1: Pairing up residues (similarity matrix)

In order to establish a match between equivalent atoms in A and B, MAMMOTH, like several other structural alignment algorithms, uses a well-established alignment technique: a similarity matrix (often inferred from and referenced as a distance matrix). A similarity matrix for alignment is an n x m array where each entry S(ai,bj) represents the pairwise similarity score between residues ai and bj. An alignment between the residues of A and B is any non-decreasing path (that is, a pair (ai,bj) in the path must appear later in the ordering of coordinates of both A and B than the preceding pair of residues in the path) from the top left corner of the array (a1,b1) to the bottom right corner (an,bm). For example the following path can be interpreted as an alignment between A = {a1, …, a11} and B = {b1, …, b8}


Any alignment can be scored by summing up the similarity scores along this path, while penalising any gaps in an appropriate way (normally, these algorithms use trial and error to decide on sensible penalties). For example, the above alignment would have the score S = S(a1,b1) + S(a2,b2) + S(a3,b3) + S(a7,b4) + S(a8,b5) + S(a9,b6) + S(a10,b7) + S(a11,b8) + α + 2β, where α and β are gap opening and gap extension penalties respectively. The optimal alignment is simply the alignment which maximises this score.

For sequence alignments similarity scores can be assigned to residues from substitution tables like BLOSUM. However, it is not immediately clear of an appropriate equivalent for structures. MAMMOTH, like several other algorithms, defines the similarity between different residues by examining their local structural landscape. Specifically, this means comparing fragments of each backbone, centred on the residue of interest. MAMMOTH uses the URMS distance between heptapeptide fragments. This distance is illustrated below using 2D chains and tripeptide fragments.


Comparing residues a2 and b3 involves looking at the directions between each successive residue of the fragment. Each vector is mapped to the unit sphere, beginning at the origin and ending at the surface of the sphere (in this case 2 vectors are considered, and in MAMMOTH’s case 6 different 3D vectors are mapped). The optimal rotation is found, superposing equivalent vectors as best as possible, and then the RMSD of the endpoints on the surface of the sphere is calculated as URMS(ai,bj).

Aside: The optimal superposition of a set of vectors is actually a non-trivial problem. It is essentially equivalent to step 2 in our alignment protocol outlined above, but is significantly easier for the 6 vectors characterising a fragment in MAMMOTH’s algorithm.

Finally, S(ai,bj) is calculated by converting the distance into a similarity measure:


where URMSR is the expected URMS of a random set of vectors and:


The optimal alignment through this MAMMOTH matrix is the path which maximises the sum of similarities between matched residues (each residue being at the centre of the heptapeptide fragment) using gap opening and extension penalties of 7.00 and 0.45 respectively.

Step 2: Global superposition (MaxSub)

The above alignment results in a match M’ optimising the local structural similarity of residues in each structure, however, their is no guarantee that this will result in a set of coordinates close in global space. In order to finalise the match set M ⊆ M’ as well as calculating the optimal superposition of the paired residues of A onto their equivalent points in B, MAMMOTH use the MaxSub algorithm. This is a very efficient algorithm (worth a read if you’re that way inclined) for calculating the maximal subset from a set of paired up atoms which are close in global space. MAMMOTH decide that close means < 4A away after superposition. They do not try to optimise a closer superposition than that but attempt to find the largest possible set of matched residues.

The MaxSub algorithm relies on the assumption (made for computational tractability) that the final subset M ⊆ M’ will have, somewhere, a set of at least four residues consecutive in M’. The algorithm then starts with every possible seed of four consecutive residues (just to illustrate the power of the assumption in reducing computational time: for a 150 residue protein there are just 147 such seeds, but over 2 million sets of four non-consecutive residues!! And it’s a pretty reasonable assumption to make as well). The MaxSub algorithm then calculates the superposition for those four matched pairs, extending the set of residues that are <4A away from their partners, recalculating the superposition using these new pairs as well, then removing any pairs which are no longer within the threshold of each other. It repeats these steps, gradually extending the set M, until the algorithm converges.

Scoring the alignment

Using the two approaches outlined above, MAMMOTH generates an alignment between the two input structures. In order to summarise the significance of this alignment, the algorithm generates the PSI score: the percentage structural identity (which is simply the size of the maximum subset divided by the length of the shortest protein). As a global measure of the strength of similarity the PSI score is poorly constructed and scales with protein length. In order to adjust for this bias, MAMMOTH fits a Gumbel distribution to PSI scores obtained from random structure comparisons between unrelated proteins at bins of different lengths. This results in a z-score measuring, instead of the PSI of an alignment, the likelihood of obtaining a PSI score as good as that by chance between any two proteins of the same lengths.

Distance matrix clustering

In Bioinformatics, we often deal with distance matrices such as:

  • Quantifying pairwise similarities between sequences
  • Structural similarity between proteins (RMSD?)
  • etc.

Next step is to study the groupings within the distance matrix using an appropriate clustering scheme. The obvious issue with most clustering methods is that you would need to specify the number of clusters beforehand (as for K-Means). Assuming that you do not know very much about the data and ‘plotting’ it is not an option, you might try non-parametric hierarchichal clustering such as linkage. The main difference between the two approaches is that using linkage you specify what the maximal distance within each cluster should be and thus the number of clusters will be adjusted accordingly. Par contre, using K-Means you do not have such a distance-guarantee within each cluster since the number of groups is predefined.

Here I will provide a short piece of python code that employs the hcluster library to perform linkage clustering.


Download hcluster, unpack it and inside the unpacked folder type:

Alternatively, if you’re not an admin on your machine type:

 Example Code

The purpose of the example bit of code is to generate a random set of points within (0,10) in the 2D space and cluster them according to user’s euclidean distance cutoff.

When I ran the code above (python [whatever you call the script].py 2.0) that’s what I got (colors correspond to clusters with ‘magenta’ being singletons):


And there is a dendogram command on the bottom of the script to see what the clustering has actually done and where it performed the cut according to your specified cutoff (colors DO NOT correspond to clusters here):



Hcluster library forms part of scipy with very useful methods for data analysis. You can modify the above code to use a variety of other hierarchichal clustering methods which you can  further explore here.