Category Archives: Talks

A short account of the talks given by the OPIG group members and their highly esteemed guests.

Slowing the progress of prion diseases

At present, the jury is still out on how prion diseases affect the body let alone how to cure them. We don’t know if amyloid plaques cause neurodegeneration or if they’re the result of it. Due to highly variable glycophosphatidylinositol (GPI) anchors, we don’t know the structure of prions. Due to their incredible resistance to proteolysis, we don’t know a simple way to destroy prions even using in an autoclave. The current recommendation[0] by the World Health Organisation includes the not so subtle: “Immerse in a pan containing 1N sodium hydroxide and heat in a gravity displacement autoclave at 121°C”.

There are several species including Water Buffalo, Horses and Dogs which are immune to prion diseases. Until relatively recently it was thought that rabbits were immune too. “Despite rabbits no longer being able to be classified as resistant to TSEs, an outbreak of ‘mad rabbit disease’ is unlikely”.[1] That being said, other than the addition of some salt bridges and additional H-bonds, we don’t know if that’s why some animals are immune.

We do know at least two species of lichen (P. sulcata and L. plumonaria) have not only discovered a way to naturally break down prions, but they’ve evolved two completely independent pathways to do so. How they accomplish this? We’re still not sure in fact, it was only last year that it was discovered that lichens may be composed of three symbiotic partnerships and not two as previously thought.[3]

With all this uncertainty, one thing is known: PrPSc, the pathogenic form of the Prion converts PrPC, the cellular form. Just preventing the production of PrPC may not be a good idea, mainly because we don’t know what it’s there for in the first place. Previous studies using PrP-knockout have shown hints that:

  • Hematopoietic stem cells express PrP on their cell membrane. PrP-null stem cells exhibit increased sensitivity to cell depletion. [4]
  • In mice, cleavage of PrP proteins in peripheral nerves causes the activation of myelin repair in Schwann Cells. Lack of PrP proteins caused demyelination in those cells. [5]
  • Mice lacking genes for PrP show altered long-term potentiation in the hippocampus. [6]
  • Prions have been indicated to play an important role in cell-cell adhesion and intracellular signalling.[7]

However, an alternative approach which bypasses most of the unknowns above is if it were possible to make off with the substrate which PrPSc uses, the progress of the disease might be slowed. A study by R Diaz-Espinoza et al. was able to show that by infecting animals with a self-replicating non-pathogenic prion disease it was possible to slow the fatal 263K scrapie agent. From their paper [8], “results show that a prophylactic inoculation of prion-infected animals with an anti-prion delays the onset of the disease and in some animals completely prevents the development of clinical symptoms and brain damage.”

[4] “Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal”. PNAS. 103 (7): 2184–9. doi:10.1073/pnas.0510577103
[5] Abbott A (2010-01-24). “Healthy prions protect nerves”. Nature. doi:10.1038/news.2010.29
[6] Maglio LE, Perez MF, Martins VR, Brentani RR, Ramirez OA (Nov 2004). “Hippocampal synaptic plasticity in mice devoid of cellular prion protein”. Brain Research. Molecular Brain Research. 131 (1-2): 58–64. doi:10.1016/j.molbrainres.2004.08.004
[7] Málaga-Trillo E, Solis GP, et al. (Mar 2009). Weissmann C, ed. “Regulation of embryonic cell adhesion by the prion protein”. PLoS Biology. 7 (3): e55. doi:10.1371/journal.pbio.1000055

Strachey Lecture – “Artificial Intelligence and the Future” by Dr. Demis Hassabis

For this week’s group meeting, some of us had the pleasure of attending a very interesting lecture by Dr. Demis Hassabis, founder of Deep Mind. Personally, I found the lecture quite thought-evoking and left the venue with a plethora of ideas sizzling in my brain. Since one of the best ways to end mental sizzlingness is by writing things down, I volunteered to write this week’s blog post in order to say my peace about yesterday’s Strachey Lecture.

Dr. Hassabis began by listing some very audacious goals: “To solve intelligence” and “To use it to make a better world”. At the end of his talk, someone in the audience asked him if he thought it was possible to achieve these goals (“to fully replicate the brain”), to which he responded with a simple there is nothing that tells us that we can’t.

After his bold introductory statement, Dr. Hassabis pressed on. For the first part of his lecture, he engaged the audience with videos and concepts of a reinforcement learning agent trained to learn and play several ATARI games. I was particularly impressed with the notion that the same agent could be used to achieve a professional level of gaming for 49 different games. Some of the videos are quite impressive and can be seen here or here. Suffice to say that their algorithm was much better at playing ATARi than I’ll ever be. It was also rather impressive to know that all the algorithm received as input was the game’s score and the pixels on the screen.

Dr. Hassabis mentioned in his lecture that games provide the ideal training ground for any form of AI. He presented several reasons for this, but the one that stuck with me was the notion that games quite often present a very simplistic and clear score. Your goal in a game is usually very well defined. You help the frog cross the road or you defeat some aliens for points. However, what I perceive to be the greatest challenge for AI is the fact that real world problems do not come with such a clear-cut, incremental score.

For instance, let us relate back to my particular scientific question: protein structure prediction. It has been suggested that much simpler algorithms such as Simulated Annealing are able to model protein structures as long as we have a perfect scoring system [Yang and Zhou, 2015]. The issue is, currently, the only way we have to define a perfect score is to use the very structure we are trying to predict (which kinda takes the whole prediction part out of the story).

Real world problems are hard. I am sure this is no news to anyone, including the scientists at Deep Mind.

During the second part of his talk, Dr. Hassabis focused on AlphaGo. AlphaGo is Deep Mind’s effort at mastering the ancient game of Go. What appealed to me in this part of the talk is the fact that Go has such a large number of possible configurations that devising an incremental score is no simple task (sounds familiar?). Yet, somehow, Deep Mind scientists were able to train their algorithm to a point where it defeated a professional Go player.

Their next challenge? In two weeks, AlphaGo will face the professional Go player with the highest number of titles in the last decade (the best player in the world?). This makes me reminiscent of when Garry Kasparov faced Deep Blue. After the talk, my fellow OPIG colleagues also seemed to be pretty excited about the outcome of the match (man vs. food computer).

Dr. Hassabis finished by saying that his career goal would be to develop AI that is capable of helping scientists tackle the big problems. From what I gather (and from my extremely biased point of view; protein structure prediction mindset), AI will only be able to achieve this goal once it is capable of coming up with its own scores for the games we present it to play with (hence developing some form of impetus). Regardless of how far we are from achieving this, at least we have a reason to cheer for AlphaGo in a couple of weeks (because hey, if you are trying to make our lives easier with clever AI, I am all up for it).

Introduction to the protein folding problem

Recently (read: this week), I had to give a presentation on my research to my college. We were informed that the audience would be non-specialist, which in fact turned out to be an understatement. For example, my presentation followed on from a discussion of the differences in the education systems of North and South Korea for the period 1949-1960. Luckily, I had tailored my entire talk to be understandable by all and devoid of all Jargon. I choose to deviate from the prescribed topic and Instead of talking about my research specifically, I choose to discuss the protein folding problem in general. Below you’ll find the script I wrote, which I feel gives a good introduction to the core problem of this field.


The protein folding problem is one of the great projects within the life sciences. Studied by vast numbers of great scientists over the last half century, with backgrounds including chemistry, physics, maths and biology, all were beaten by the sheer complexity of the problem. As a community we still have only scraped the surface with regards to solving it. While I could, like many of you here, go into my own research in great detail and bore you wholeheartedly for the next 10 minutes with technical details and cryptic terminology, I will instead try to give an overview of the problem and why thousands of scientists around the globe are still working on cracking this.

First of all, I guess that a few of you are trying to remind yourself what a protein is; the horror that is high school biology crawling back from that area in your brain you keep for traumatic experiences like family gatherings. Luckily, I’m fairly new to the topic myself, my background being in physics and chemistry, so hopefully my explanation will be still in the naive terms that I use to explain the core concepts to myself. Proteins are the micro-machines of your body, the cogs that keep the wheels turning, the screws that hold the pieces together and the pieces themselves. Proteins run nearly all aspects of your body and biochemistry, your immune system, your digestion and your heart beating. There are approximately between 20 to 30 thousand different proteins in your body, depending on who you ask, and trillions overall. In fact, if we take every protein in our body and scale it up to the size of a penny, the proteins in a single human, albeit a rather dead human, would be enough to fill the entire pacific ocean. Basically, there is a hell of a lot of proteins, with a vast range of different types, each of which is very individual, both in its compositions and function, and, crucially, they are nearly all essential. The loss of any protein can lead to dramatic consequences including heart disease, cancer, and even death.

So now that you know that they are important and there are lots of them, what exactly is a protein? The easiest analogy I have is that of a pearl necklace, a long string of beads in a chain. Now consider your significant other has gone slightly insane and instead of purchasing jewelry for you that consists of a single bead type, or even two if you have slightly exotic tastes, they have been shopping at one of the jewellery stores found in the part of town that smells rather “herby”. You receive a necklace which has different beads across the entire length of the necklace. We have blues, yellows, pinks, and so on and so forth. In fact, we have 20 different types of beads, each with its own colour. This is basically a protein chain: each of the beads represents one of the twenty essential amino acids, each of which has its own chemical and physical properties. Now suppose you can string those pearls together in any order: red, green, blue, blue, pink etc. It turns out that the specific order that these beads are arranged along the length of the protein chain define exactly how this chain “crumples” into a 3D shape. If you think that adding an extra dimension is impossible, just consider crumpling a piece of paper; that is 2D –> 3D transition (mathematicians please bite your tongue). Now one string of colours, blue, blue, pink for example, will crunch into one shape, and that shape may become your muscle, while a different sequence, say, green, blue, orange, will crumple down into something different, for example an antibody to patrol your blood stream.

So essentially we have this “genetic code”, the sequence of amino acids (or beads), which in turn defines the shape that the protein will take. We in fact know that it is this shape that is the most important aspect of any protein, this having been found to define the protein’s actual function. This is because, returning to the bead analogy, we can change up to 80% of the beads to different colour while still retaining the same shape and function. This is amazing when you consider how many other objects can have their baseline composition changed to the same extent while still retaining the same function. The humble sausage is one of those objects (actually below 40% meat content they are referred to as “bangers”), but even then would you want 80% of you sausage to be filler. There is a reason Tesco value sausages taste so different to the nice ones you buy at the butchers. Returning to proteins, we are not trying to say that the sequence isn’t important, sometimes changing just a single bead can lead to a completely different shape. Instead it says that the shape is the critical aspect which defines the function. To summarise, sequence leads to shape, which in turn leads to function.

This is unfortunate, because while it is getting increasingly simple to experimentally determine the sequence of a protein, that is the exact order of coloured beads, the cost and time of getting the corresponding structure (shape) is still extremely prohibitive. In fact, we can look at two of the major respective databases, the PDB, which contains all known proteins structures, and GENBANK, which contains all known protein sequences, and compare the respective number of entries. The disparity between the two is huge, we are talking orders of magnitude huge, 10^15 huge, i.e the number of humans on the planet squared huge. AND this gap is growing larger every year. Basically, people in the last few years have suddenly gained access to cheap and fast tools to get a protein’s sequence, to the extent that people are widely taking scoops of water across the world and sequencing everything, not even bothering to separate the cells and microorganisms beforehand. Nothing analogous exists to get the structure of a protein. The process taking months to years, depending on many factors, each of which may be “something” for one protein and then a completely different “something” for a similar protein. This has led to a scenario where we know the sequence of every protein in the human genome, yet we only know the structure of only about 10% of them. This is utterly preposterous in my opinion given how important this information is to us! We basically don’t know what 90% of our DNA does!

Basically, until an analogous method for structure determination is produced, we have no choice but to turn to predictive methods to suggest the function of proteins that we do not have the structure for. This is important as it allows us, to some degree, target proteins that we “think” may have an important effect. If we didn’t do this, we simply would be searching for needles in haystacks. This is where my research, and that of my group, kicks in. We attempt to take these sequences, these string of beads, and predict the shape that they produce. Unfortunately, the scientific community as a whole still relatively sucks at this. Currently, we are only successful in predicting structures for very small proteins, and when anything more complex is attempted, we, in general, fail utterly miserably. In my opinion, this is because the human body is by far the most complex system on the planet and so far we have tried to simply supplant physics on top of the problem. This has failed miserably due to the sheer complexity and multitude of factors involved. Physics has mostly nice vacuums and pleasant equations, however ask a physicist about a many body system and they will cry. So many factors are involved that we must integrate them altogether which is why there are so many people are working on this, and will be for many years to come. Well, I guess that’s good news for my future academic career.

Anyway, I hope this talk has given you some degree of insight into the work I do and you have learned something about how your body works. For those extremely interested, please feel free to approach me later and I will happily regale you with the exact aspect of protein folding I work on. But for now I would love to try and answer any questions you all have on the content contained in this talk.

Journal club: Half a century of Ramachandran plots

In last week’s journal club we delved into the history of Ramachandran plots (Half a century of Ramachandran plots; Carugo & Djinovic-Carugo, 2013).

Polypeptide backbone dihedral angles

Polypeptide backbone dihedral angles. Source: Wikimedia Commons, Bensaccount

50 years ago Gopalasamudram Narayana Ramachandran et al. predicted the theoretically possible conformations of a polypeptide backbone. The backbone confirmations can be described using three dihedral angles: ω, φ and ψ (shown to the right).

The first angle, ω, is restrained to either about 0° (cis) or about 180° (trans) due to the partial double bond character of the C-N bond. The φ and ψ angles are more interesting, and the Ramachandran plot of a protein is obtained by plotting φ/ψ angles of all residues in a scatter plot.

The original Ramachandran plot showed the allowed conformations of the model compound N-acetyl-L-alanine-methylamide using a hard-sphere atomic model to keep calculations simple. By using two different van der Waals radii for each element positions on the Ramachandran plot could be classified into either allowed regions, regions with moderate clashes and disallowed regions (see Figure 3 (a) in the paper).

The model compound does not take side chains into account, but it does assume that there is a side chain. The resulting Ramachandran plot therefore does not describe the possible φ/ψ angles for Glycine residues, where many more conformations are plausible. On the other end of the spectrum are Proline residues. These have a much more restricted range of possible φ/ψ angles. The φ/ψ distributions of GLY and PRO residues are therefore best described in their own Ramachandran plots (Figure 4 in the paper).

Over time the Ramachandran plot was improved in a number of ways. Instead of relying on theoretical calculations using a model compound, we can now rely on experimental observations by using high quality, hand picked data from the PDB. The way how the Ramachandran plot is calculated has also changed: It can now be seen as a two-dimensional, continuous probability distribution, and can be estimated using a full range of smoothing functions, kernel functions, Fourier series and other models.
The modern Ramachandran plot is much more resolved than the original plot. We now distinguish between a number of well-defined, different regions which correlate with secondary protein structure motifs.

Ramachandran plots are routinely used for structure validation. The inherent circular argument (A good structure does not violate the Ramachandran plot; The plot is obtained by looking at the dihedral angles of good structures) sounds more daring than it actually is. The plot has changed over time, so it is not as self-reinforcing as one might fear. The Ramachandran plot is also not the ultimate guideline. If a new structure is found that claims to violate the Ramachandran plot (which is based on a huge body of cumulative evidence), then this claim needs to be backed up by very good evidence. A low number of violations of the plot can usually be justified. The Ramachandran plot is a local measure. It therefore does not take into account that domains of a protein can exert a force on a few residues and just ‘crunch’ it into an unusual conformation.

The paper closes with a discussion of possible future applications and extensions, such as the distribution of a protein average φ/ψ and an appreciation of modern web-based software and databases that make use of or provide insightful analyses of Ramachandran plots.

Viewing ligands in twilight electron density

In this week’s journal club we discussed an excellent review paper by E. Pozharski, C. X. Weichenberger and B. Rupp investigating crystallographic approaches to protein-ligand complex elucidation. The paper assessed and highlighted the shortcomings of deposited PDB structures containing ligand-protein complexes. It then made suggestions for the community as a whole and for researchers making use of ligand-protein complexes in their work.

The paper discussed:

  • The difficulties in protein ligand complex elucidation
  • The tools to assess the quality of protein-ligand structures both qualitative and quantitative
  • The methods used describing their analysis of certain PDB structures
  • Some case studies visually demonstrating these issues
  • Some practical conclusions for the crystallographic community
  • Some practical conclusions for non-crystallographer users of protein-ligand complex structures from the PDB

The basic difficulties of ligand-protein complex elucidation

  • Ligands have less than 100% occupancy – sometimes significantly less and thus will inherently show up less clearly in the overall electron density.
  • Ligands make small contributions to the overall structure and thus global quality measures , such as r-factors, will be affected only minutely by the ligand portion of the structure being wrong
  • The original basis model needs to be used appropriately. The r-free data from the original APO model should be used to avoid model bias

The following are the tools available to inspect the quality of agreement between protein structures and their associated data.

  • Visual inspection of the Fo-Fc and 2Fo-Fc maps,using software such as COOT, is essential to assess qualitatively whether a structure is justified by the evidence.
  • Use of local measures of quality for example real space correlation coefficients (RSCC)
  • Their own tool, making use of the above as well as global quality measure resolution

Methods and results

In a separate publication they had analysed the entirety of the PDB containing both ligands and published structure factors. In this sample they demonstrate 7.6% had RSCC values of less than 0.6 the arbitrary cut off they use to determine whether the experimental evidence supports the model coordinates.

Figure to show an incorrectly oriented ligand (a) and its correction (b)

An incorrectly oriented ligand (a) and its correction (b). In all of these figures Blue is the 2mFoDFc map contoured at 1σ and Green and Red are positive and negative conturing of the mFoDFc map at 3σ

In this publication they visually inspected a subset of structures to assess in more detail how effective that arbitrary cutoff is and ascertain the reason for poor correlation. They showed the following:

(i) Ligands incorrectly identified as questionable,false positives(7.4%)
(ii) Incorrectly modelled ligands (5.2%)
(iii) Ligands with partially missing density (29.2%).
(iv) Glycosylation sites (31.3%)
(v) Ligands placed into electron density that is likely to
originate from mother-liquor components
(vi) Incorrect ligand (4.7%)
(vii) Ligands that are entirely unjustified by the electron
density (11.9%).

The first point on the above data is that the false-positive rate using RSCC of 0.6 is 7.4%. This demonstrates that this value is not sufficient to accurately determine incorrect ligand coordinates. Within the other categories all errors can be attributed to one of or a combination of the following two factors:

  • The inexperience of the crystallographer being unable to understand the data in front of them
  • The wilful denial of the data in front of the crystallographer in order that they present the data they wanted to see
Figure to show a ligand placed in density for a sulphate ion from the mother liquor (a) and it's correction (b)

A ligand incorrectly placed in density for a sulphate ion from the mother liquor (a) and it’s correction (b)

The paper observed that a disproportionate amount of poor answers was derived from glycosylation sites. In some instances these observations were used to inform the biochemistry of the protein in question. Interestingly this follows observations from almost a decade ago, however many of the examples in the Twilight paper were taken from 2008 or later. This indicates the community as a whole is not reacting to this problem and needs further prodding.

Figure to show an incomplete glycosylation site inaccurately modeled

Figure to show an incomplete glycosylation site inaccurately modeled

Conclusions and suggestions

For inexperienced users looking at ligand-protein complexes from the PDB:

  • Inspect the electron density map using COOT if is available to determine qualitatively is their evidence for the ligand being there
  • If using large numbers of ligand-protein complexes, use a script such as Twilight to find the RSCC value for the ligand to give some confidence a ligand is actually present as stated

For the crystallographic community:

  • Improved training of crystallographers to ensure errors due to genuine misinterpretation of the underlying data are minimised
  • More submission of electron-density maps, even if not publically available they should form part of initial structure validation
  • Software is easy to use but difficult to analyse the output

GPGPUs for bioinformatics

As the clock speed in computer Central Processing Units (CPUs) began to plateau, their data and task parallelism was expanded to compensate. These days (2013) it is not uncommon to find upwards of a dozen processing cores on a single CPU and each core capable of performing 8 calculations as a single operation. Graphics Processing Units were originally intended to assist CPUs by providing hardware optimised to speed up rendering highly parallel graphical data into a frame buffer. As graphical models became more complex, it became difficult to provide a single piece of hardware which implemented an optimised design for every model and every calculation the end user may desire. Instead, GPU designs evolved to be more readily programmable and exhibit greater parallelism. Top-end GPUs are now equipped with over 2,500 simple cores and have their own CUDA or OpenCL programming languages. This new found programmability allowed users the freedom to take non-graphics tasks which would otherwise have saturated a CPU for days and to run them on the highly parallel hardware of the GPU. This technique proved so effective for certain tasks that GPU manufacturers have since begun to tweak their architectures to be suitable not just for graphics processing but also for more general purpose tasks, thus beginning the evolution General Purpose Graphics Processing Unit (GPGPU).

Improvements in data capture and model generation have caused an explosion in the amount of bioinformatic data which is now available. Data which is increasing in volume faster than CPUs are increasing in either speed or parallelism. An example of this can be found here, which displays a graph of the number of proteins stored in the Protein Data Bank per year. To process this vast volume of data, many of the common tools for structure prediction, sequence analysis, molecular dynamics and so forth have now been ported to the GPGPU. The following tools are now GPGPU enabled and offer significant speed-up compared to their CPU-based counterparts:

Application Description Expected Speed Up Multi-GPU Support
Abalone Models molecular dynamics of biopolymers for simulations of proteins, DNA and ligands 4-29x No
ACEMD GPU simulation of molecular mechanics force fields, implicit and explicit solvent 160 ns/day GPU version only Yes
AMBER Suite of programs to simulate molecular dynamics on biomolecule 89.44 ns/day JAC NVE Yes
BarraCUDA Sequence mapping software 6-10x Yes
CUDASW++ Open source software for Smith-Waterman protein database searches on GPUs 10-50x Yes
CUDA-BLASTP Accelerates NCBI BLAST for scanning protein sequence databases 10 Yes
CUSHAW Parallelized short read aligner 10x Yes
DL-POLY Simulate macromolecules, polymers, ionic systems, etc on a distributed memory parallel computer 4x Yes
GPU-BLAST Local search with fast k-tuple heuristic 3-4x No
GROMACS Simulation of biochemical molecules with complicated bond interactions 165 ns/Day DHFR No
GPU-HMMER Parallelized local and global search with profile Hidden Markov models 60-100x Yes
HOOMD-Blue Particle dynamics package written from the ground up for GPUs 2x Yes
LAMMPS Classical molecular dynamics package 3-18x Yes
mCUDA-MEME Ultrafast scalable motif discovery algorithm based on MEME 4-10x Yes
MUMmerGPU An open-source high-throughput parallel pairwise local sequence alignment program 13x No
NAMD Designed for high-performance simulation of large molecular systems 6.44 ns/days STMV 585x 2050s Yes
OpenMM Library and application for molecular dynamics for HPC with GPUs Implicit: 127-213 ns/day; Explicit: 18-55 ns/day DHFR Yes
SeqNFind A commercial GPU Accelerated Sequence Analysis Toolset 400x Yes
TeraChem A general purpose quantum chemistry package 7-50x Yes
UGENE Opensource Smith-Waterman for SSE/CUDA, Suffix array based repeats finder and dotplot 6-8x Yes
WideLM Fits numerous linear models to a fixed design and response 150x Yes

It is important to note however, that due to how GPGPUs handle floating point arithmetic compared to CPUs, results can and will differ between architectures, making a direct comparison impossible. Instead, interval arithmetic may be useful to sanity-check the results generated on the GPU are consistent with those from a CPU based system.

Journal club: Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature

In the last journal club we covered the paper Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature (Kern et al., 2013), currently still in Science Express.

Structure of Photosystem II

Structure of Photosystem II, PDB 2AXT
CC BY-SA 3.0 Curtis Neveu

This paper describes an experiment on the Photosystem II (PSII) protein complex. PSII is a large protein complex consisting of about 20 subunits with a combined weight of ca. 350 kDa. As its name suggests, this complex plays a crucial role in photosynthesis: it is responsible for the oxidation (“splitting up”) of water.

In the actual experiment (see the top right corner of Figure 1 in the paper) three experimental methods are combined: PSII microcrystals (5-15µm) are injected from the top into the path of an X-ray pulse (blue). Simultaneously, an emission spectrum is recorded (yellow, detector at the bottom). And finally in a separate run the injected crystals are treated (‘pumped’) with a visible laser (red) before they hit the X-ray pulse.

Let’s take a look at each of those three in a little more detail.

X-ray diffraction (XRD)

In a standard macromolecular X-ray crystallography experiment a crystal containing identical molecules of interest (protein) at regular, ordered lattice points is exposed to an X-ray beam. Some X-rays are elastically scattered and cause a diffraction pattern to form on a detector. By analysing the diffraction patterns of a rotating crystal it is possible to calculate the electron density distribution of the molecule in question, and thus determine its three dimensional structure.

An intense X-ray beam however also systematically damages the sample (Garman, 2010). For experiments using in-house X-ray generators or synchrotrons it is therefore recommended not to exceed a total dose of 30 MGy on any one crystal (Owen et al., 2006).

Aerial view of the LCLS site. The accelerator is 3.2 kilometres long.

The experiment described in the current paper however was not conducted using a run-of-the-mill synchrotron, but with the Linac Coherent Light Source (LCLS), an X-ray free electron laser. Here each diffraction image results from an individual crystal exposed to a single X-ray pulse of about 50 femtoseconds, resulting in a peak dose of ~150 MGy. Delivering these extreme doses in very short X-ray pulses lead to the destruction of the sample via a coulomb explosion.

As the sample is destroyed only one diffraction image can be taken per crystal. This causes two complications: Firstly, a large number of sample crystals are required. This explains why the experiment required a total beam time of 6 hours and involved over 2.5 million X-ray shots and processing an equal number of diffraction images. Secondly, the resulting set of (usable) diffraction images are an unordered, random sampling of the crystal orientation space. This presents a computational challenge unique to XFEL setups: before the diffraction data can be integrated, the orientation of the crystal lattice needs to be determined for each individual diffraction image.

X-ray diffraction allows us to obtain a electron density map of the entire unit cell, and therefore the entire crystal. But we can only see ordered areas of the crystal. To really see small molecules in solvent channels, or the conformations of side chains on the protein surface they need to occupy the same positions within each unit cell. For most small molecules this is not the case. This is why you will basically never see compounds like PEGs or Glycerol in your electron density maps, even though you used them during sample preparation. For heavy metal compounds this is especially annoying. They disproportionately increase the X-ray absorption (Holton, 2009; with a handy table 1) and therefore shorten the crystal’s lifetime. But they do not contribute to the diffraction pattern (And that is why you should back-soak; Garman & Murray, 2003).

X-ray emission spectroscopy (XES)

PSII contains a manganese cluster (Mn4CaO5). This cluster, as with all protein metallocentres, is known to be highly radiation sensitive (Yano et al., 2005). Apparently at a dose of about 0.75 MGy the cluster is reduced in 50% of the irradiated unit cells.

Diffraction patterns represent a space- and time-average of the electron density. It is very difficult to quantify the amount of reduction from the obtained diffraction patterns. There is however a better and more direct way of measuring the states of the metal atoms: X-ray emission spectroscopy.

Basically the very same events that cause radiation damage also cause X-ray emissions with very specific energies, characteristic for the involved atoms and their oxidation state. An MnIVO2 energy spectrum looks markedly different to an MnIIO spectrum (bottom right corner of Figure 1).

The measurements of XRD and XES are taken simultaneously. But, differently to XRD, XES does not rely on crystalline order. It makes no difference whether the metallocentres move within the unit cell as a result of specific radiation damage. Even the destruction of the sample is not an issue: we can assume that XES will record the state of the clusters regardless of the state of the crystal lattice – up to the point where the whole sample blows up. But at that point we know that due to the loss of long-range order the X-ray diffraction pattern will no longer be affected.

The measurements of XES therefore give us a worst-case scenario of the state of the manganese cluster in the time-frame where we obtained the X-ray diffraction data.

Induced protein state transition with visible laser

Another neat technique used in this experiment is the activation of the protein by a visible laser in the flight path of the crystals as they approach the X-ray interaction zone. With a known crystal injection speed and a known distance between the visible laser and the X-ray pulse it becomes possible to observe time-dependent protein processes (top left corner of Figure 1).

What makes this paper special? (And why is it important to my work?)

It has been theorized and partially known that the collection of diffraction data using X-ray free electron lasers outruns traditional radiation damage processes. Yet there was no conclusive proof – up until now.

This paper has shown that the XES spectra of the highly sensitive metallocentres do not indicate any measurable (let alone significant) change from the idealized intact state (Figure 3). Apparently the highly sensitive metallocentres of the protein stay intact up to the point of complete sample destruction, and certainly well beyond the point of sample diffraction.

Or to put another way: even though the crystalline sample itself is destroyed under extreme conditions, the obtained diffraction pattern is – for all intents and purposes – that of an intact, pristine, zero dose state of the crystal.

This is an intriguing result for people interested in macromolecular crystallography radiation damage. In a regime of absurdly high doses, where the entire concept of a ‘dose’ for a crystal breaks down (again), we can obtain structural data with no specific radiation damage present at the metallocentres. As metallocentres are more susceptible to specific radiation damage it stands to reason that the diffraction data may be free of all specific damage artefacts.

It is thought that most deposited PDB protein structures containing metallocentres are in a reduced state. XFELs are being constructed all over the world and will be the next big thing in macromolecular crystallography. And now it seems that the future of metalloproteins got even brighter.

Journal Club: The complexity of Binding

Molecular recognition is the mechanism by which two or more molecules come together to form a specific complex. But how do molecules recognise and interact with each other?

In the TIBS Opinion article by Ruth Nussinov group, an extended conformational selection model is described. This model includes the classical lock-and-key, induced fit, conformational selection mechanisms and their combination.

The general concept of equilibrium shift of the ensemble was proposed nearly 15 years ago, or perharps earlier. The basic idea is that proteins in solution pre-exist in a number of conformational substates, including those with binding sites complementary to a ligand. The distribution of the substates can be visualised as free energy landscape (see figure above), which helps in understanding the dynamic nature of the conformational equilibrium.

This equilibrium is not static, it is sensitive to the environment and many other factors. An equilibrium shift can be achieved by (a) sequence modifications of special protein regions termed protein segments, (b) post-translational modifications of a protein, (c) ligand binding, etc.

So why are these concepts discussed and published again?

While the theory is straight-forward, proving conformational selection is hard and it is even harder to quantify it computationally. Experimental techniques such Nuclear Magnetic Resonance (NMR), single molecule studies (e.g. protein yoga), targeted mutagenesis and its effect on the energy landscape, plus molecular dynamics (MD) simulations have been helping to conceptualise conformational transitions. Meanwhile, there is still a long way to go before a full understanding of atomic scale pathways is achieved.

Talk: Membrane Protein 3D Structure Prediction & Loop Modelling in X-ray Crystallography

Seb gave a talk at the Oxford Structural Genomics Consortium on Wednesday 9 Jan 2013. The talk mentioned the work of several other OPIG members. Below is the gist of it.

Membrane protein modelling pipeline

Homology modelling pipeline with several membrane-protein-specific steps. Input is the target protein’s sequence, output is the finished 3D model.

Fragment-based loop modelling pipeline for X-ray crystallography

Given an incomplete model of a protein, as well as the current electron density map, we apply our loop modelling method FREAD to fill in a gap with many decoy structures. These decoys are then scored using electron density quality measures computed by EDSTATS. This process can be iterated to arrive at a complete model.

Over the past five years the Oxford Protein Informatics Group has produced several pieces of software to model various aspects of membrane protein structure. iMembrane predicts how a given protein structure sits in the lipid bilayer. MP-T aligns a target protein’s sequence to an iMembrane-annotated template structure. MEDELLER produces an accurate core model of the target, based on this target-template alignment. FREAD then fills in the remaining gaps through fragment-based loop modelling. We have assembled all these pieces of software into a single pipeline, which will be released to the public shortly. In the future, further refinements will be added to account for errors in the core model, such as helix kinks and twists.

X-ray crystallography is the most prevalent way to obtain a protein’s 3D structure. In difficult cases, such as membrane proteins, often only low resolution data can be obtained from such experiments, making the subsequent computational steps to arrive at a complete 3D model that much harder. This usually involves tedious manual building of individual residues and much trial and error. In addition, some regions of the protein (such as disordered loops) simply are not represented by the electron density at all and it is difficult to distinguish these from areas that simply require a lot of work to build. To alleviate some of these problems, we are developing a scoring scheme to attach an absolute quality measure to each residue being built by our loop modelling method FREAD, with a view towards automating protein structure solution at low resolution. This work is being carried out in collaboration with Frank von Delft’s Protein Crystallography group at the Oxford Structural Genomics Consortium.