Foldit's 10 Year Anniversary!
Today marks the 10-year anniversary of Foldit’s launch on May 9, 2008!
In the past decade, Foldit players have advanced protein science by accurately predicting the structure of a viral protein1, by developing an algorithm for protein modeling2, and by redesigning a protein enzyme with improved activity3. Foldit players have shown that they can refine protein models better than sophisticated computer programs4, and that they can interpret electron density maps as well as expert crystallographers5. We have high hopes for the next 10 years of Foldit, and can't wait to see what Foldit players will discover next!
Protein Design in Foldit
Most recently, Foldit players have been designing brand new proteins from scratch. The ability to design proteins is a big milestone for Foldit players, and we’re excited about the new types of problems that we can start to tackle with protein design in Foldit! This achievement has been a long time in the making—below you can review previous blog posts to follow this progress over the last four years. Play the latest design puzzle now!
Nov. 1, 2013 - First batch of Foldit player-designed proteins selected for testing
Mar. 25, 2014 - Improvements in Foldit player-designed proteins
Jun. 18, 2014 - First positive testing results for a Foldit player-designed protein
Feb. 10, 2015 - First alpha/beta Foldit designs selected for testing
Feb. 28, 2017 - Better backbones yield promising alpha/beta designs
Mar. 1, 2017 - Diverse player designs fold up in the wet lab
Apr. 15, 2017 - Protein crystallography of a Foldit player design
May 30, 2017 - X-ray diffraction of a protein crystal
A high-resolution crystal structure (cyan) aligned with the design model (green) shows that this protein folds up just as it was designed by Waya, Galaxie, and Susume. The protein backbone aligns to the design with a Cα RMSD of 1.1 Å, and the sidechains in the protein core pack just as intended.
Small Molecule Design in Foldit
We’re also excited to ramp up small-molecule design in Foldit, allowing Foldit players to create new ligands that could bind to protein targets! Play the latest small-molecule design puzzle now!
New tools allow Foldit players to build small molecules that can bind to protein targets
We'd like to thank all the Foldit players that have contributed to Foldit over the last 10 years! None of this would have been possible without you! Happy folding!
1. Khatib, F. et al. Crystal structure of a monomeric retroviral protease solved by protein folding game players. Nat Struct Mol Biol 18, 1175–1177 (2011).
2. Khatib, F. F. et al. Algorithm discovery by protein folding game players. Proc Natl Acad Sci U S A 108, 18949–18953 (2011).
3. Eiben, C. B. et al. Increased Diels-Alderase activity through backbone remodeling guided by Foldit players. Nature Biotechnology 30, 190–192 (2012).
4. Cooper, S. et al. Predicting protein structures with a multiplayer online game. Nature 466, 756–760 (2010).
5. Horowitz, S. et al. Determining crystal structures through crowdsourcing and coursework. Nat Commun 7, 12549 (2016).
Aflatoxin Challenge Update
Foldit Community, we are ready for an Aflatoxin update! Thank you for all your efforts in designing the first round of designs to combat aflatoxin. To date we have had hundreds of players design >400,000 structures! There is a lot to choose from and it has been a fantastic way to start off this effort. Players have been incredibly creative, and have engineered exciting new molecular interactions that truly have the potential to stabilize the aflatoxin hydrolysis transition state we provided in the models. Two examples are below: the first is a pi-pi stacking interaction between a tryptophan and the conjugated ring system of aflatoxin AFB1 (left, Figure 1). If the designed protein is stable enough in this state to provide this interaction in the physical world it would almost certainly position aflatoxin in a manner poised for enzymatic hydrolysis. The second interaction is a classical hydrogen bond with a ketone group (right, Figure 1). Again, these energetically favorable and strong interactions will provide the physical properties needed to stabilize aflatoxin AFB1 in a manner ready for hydrolysis by the naturally occurring catalytic core of the enzymes. We look forward to seeing more of these interactions in subsequent rounds!
Figure 1. AFB1 interactions in Foldit player designs. In both images the white structure is the native enzyme (starting structure), and the green is a Foldit player design. Left, a double mutation accommodated by some backbone movement enabled a pi-stacking interaction between tryptophan and AFB1. Right, a single mutation and backbone change introduce a hydrogen bond with the ketone group on AFB1. Keep making changes! And remember that it is not only important to interact with AFB1, but also to stabilize the new protein structure to reinforce the AFB1 interactions!
On the experimental front we are excited to announce that we have transferred all methods into a microtiter plate format and have tested the first 100 GeneStrings from ThermoFisher designed by players (Figure 2). These were selected on a variety of factors (~30 of the top scoring based on overall score; ~30 based on top AFB1 energy with above average overall score; best score of 20 players sorted on best scoring designs; all of the player Scientist Shares). While the process from design to data was seamless, unfortunately all of the data was negative (i.e. none of the designs degraded aflatoxin). We are going back to this first round and re-evaluating how we picked designs, as well as going back and refining some of the designs we thought were most interesting ourselves, however in the meantime we want to get another round of puzzles going. Don’t lose hope! We expect this will require several rounds of design as we optimize the puzzle and solution selection parameters, as well as start to prepare a few new scaffold proteins to try. But we are confident we will find something in the next few rounds and we appreciate your diligence and efforts in helping solve this global issue!
There was great feedback from the community about the original puzzles and we plan to adjust the next puzzles based on this feedback. Most importantly, we plan to trim some of the frozen protein regions, so you can deal with a smaller puzzle and focus on the regions of interest. We may also upweight the ligand scoring, to encourage more interactions with the ligand. Although some players have requested the ability to move the ligand around the binding pocket, we will continue to keep the ligand fixed in place; the ligand's orientation with respect to the catalytic residues is critical if we want the reaction to occur!
We will continue to update you as we go, but we're off to a strong start! Check out Puzzle 1497: Aflatoxin Challenge: Round 5 now!( Posted by 82 2278 | Wed, 03/14/2018 - 23:04 | 6 comments )
New Update in 'Experimental' Update Group!
As many of you are probably aware, Rosetta is the "science engine" behind Foldit. It's been about two years since we last updated the version of Rosetta that Foldit uses under the hood. Since then, there have been many developments and improvements in Rosetta, and we thought it's time to update again.
Here's just a sampling of some of the improvements that come with the updated Rosetta:
* Fixes to some of the random crashes on MacOS
* Fixes to electron density-related crashes on Linux
* Fixes to symmetry-related crashes on all platforms
* Better support for non-protein residues, such as RNA, carbohydrates, lipids and non-canonical amino acids
* Support for modeling membrane proteins
* Improved detection of native-like hydrogen bonding networks
But perhaps the biggest change that comes with the update is improvements to scoring. There's been a *lot* of work put into the Rosetta scoring function recently, and just about every portion of scoring has been re-evaluated and re-optimized. (For those who want a nitty-gritty breakdown of the changes, a comprehensive overview has been published, along with details on how things were optimized.)
Here's a demonstration of the improvement. When looking at the ability to discriminate native-like proteins from non-native-like ones, for many proteins the new scoring function is able to do a much better job than the older scoring function:
Here each red point represents a structure prediction run for a different protein. The discrimination ability of the two scoring functions are plotted, using a metric where 0.0 represents no discrimination between native-like and non-native, and 1.0 represents ideal discrimination. The diagonal line represents no difference between the two scoring functions, and any points above the diagonal line represent proteins where the new scoring function does a better job than the old one.
This also is reflected in the score-versus-rmsd "funnel" plots for the predictions, where the new scoring function does a better job of eliminating false minima (blue) than the older score function does. (In these plots, better scoring structures are lower on the y-axis, and more native-like structures are further to the left. Eliminating false minima means a selection of top-scoring structures is less likely to include non-native-like ones.)
This improvement isn't limited only to protein structure prediction. The new scoring function shows discrimination improvements in a wide range of protein prediction problems, including protein-protein and protein-small-molecule interaction predictions.
There is a slight drawback to these improvements, though. The new scoring function is slower than the current one. (In our tests, it averages about 30% slower.) We don't anticipate this being noticeable in general interactive use, but it may affect things like long-running shakes and wiggles. Most affected will be scripts which use a set number of iterations of shake and wiggle - these will run for longer, and if you've optimized the number of iterations for the current scoring function, the optimal number of iterations may have changed in the new one.
We're excited about these score function improvements, though, and think the better results are worth the slowdown. You might spend a bit more time working on a single structure, but you should hopefully spend much less time working on "bad" or "scientifically uninteresting" structures.
So, if you're feeling adventurous, please help us out by testing the updated version. To do this, switch your update group to `experimental`. In addition to testing how the slowdown affects scripts, we also want to make sure no bugs have slipped in on how the various tools behave. -- Note that, due to the difference in scoring, the puzzles available with `experimental` are not the same as with the `main` and `devprev` clients. If you want to play the regular Foldit puzzles, you'll need to switch your update group back to 'main'. None of the 'experimental' puzzles will count towards your website rank, but they will help us work out any issues prior to releasing it to the general public!( Posted by rmoretti 82 899 | Thu, 02/08/2018 - 21:42 | 3 comments )
Aflatoxin: a cancer-causing glue
In our previous blog post, we announced our Aflatoxin Challenge – a new series of Foldit puzzles designed to tackle a common poison known as aflatoxin. This week, Baker Lab scientist ianh offers a more detailed picture of the chemistry behind aflatoxin's harmful effects.
Most people on Earth consume aflatoxins every day. Aflatoxins are compounds produced by certain fungi that can grow in or on almost all grains and groundnuts. Aflatoxins are known hepatocarcinogens, meaning they cause liver cancer in high doses. Liver cancer is the third leading cause of cancer death globally, with 83% of cases occurring in East Asia and sub-Saharan Africa where aflatoxin exposure is highest.
What makes aflatoxin so toxic?
Surprisingly, aflatoxin itself isn’t toxic. Once ingested, our body uses its normal metabolic processes to try to break it down. It turns out a metabolic product of aflatoxin – not aflatoxin itself – is harmful, and the chemistry behind its toxicity is both frightening and familiar.
One of the metabolic enzymes that acts on aflatoxin is CYP3A4, a vitally import liver enzyme tasked with breaking down different complex molecules. Its normal targets are molecules produced by our own bodies, like the sex hormones testosterone and estrogen, but CYP3A4 can also safely chew up some of the complex chemicals we put in our body, such as caffeine (found in coffee and tea) and lidocaine (a local anesthetic commonly used by dentists).
When CYP3A4 metabolizes a chemical it changes that molecule’s structure in some way. When it acts on aflatoxin, it adds a chemical feature – chemists call this new feature an epoxide group.
Epoxide groups are highly reactive, meaning they are unstable on their own and like to bond with other nearby chemicals. This extreme reactivity can be harnessed for many practical applications, including adhesion, electrical insulation, and industrial manufacturing. Some of the strongest glues ever made – epoxy glues – are based on epoxide chemistry.
Once CYP3A4 converts aflatoxin into epoxy-aflatoxin, the compound doesn’t wait around for long. It quickly reacts with other chemicals in our cells, especially amines.
One of the best places to find amines in our cells in our DNA. Each of the four letters of DNA – A, T, G, and C – is an amine. Epoxy-aflatoxin reacts especially strongly with guanosine (G), forming a nearly-unbreakable bond. This permanently damages DNA.
DNA damage is problematic, not only because it interferes with the natural processes of the damaged cell, but because this damage can be passed on to descendants when the damaged cell replicates. In this way, a single error in a single cell may be amplified over time to affect a large population of cells.
The liver is especially prone to this cascade because it (1) expresses high levels of the CYP3A4 protein that metabolizes aflatoxin, and (2) because liver cells reproduce at faster rates than cells of other organs, allowing DNA mutations to accumulate and propagate much more quickly.
Human DNA includes a gene that encodes for the tumor-suppressing protein p53. The normal role of the p53 protein is to police DNA damage in the cell. It recognizes DNA damage and can initiate DNA repair processes or, in extreme cases, induce cell death. Without functional p53 protein, DNA damage runs rampant in a cell. Dysfunctional p53 is strongly associated with many forms of cancer in humans.
It seems that one particular guanosine in the p53 gene is especially susceptible to aflatoxin damage. Damage to this guanosine is propagated to descendant cells as a G -> T mutation in the p53 gene; the G -> T mutation in the p53 gene results in a ARG -> SER mutation in the p53 protein. The p53 protein falls victim to the very type of DNA damage it is meant to avert! This mutated version of p53 is exceedingly common in liver cancer patients.
In summary: aflatoxin causes DNA damage in the liver, resulting in a population of cells with defective p53 and prompting tumor growth.
Help us design a protein to break down this toxic compound by playing the latest Foldit puzzle, 1445: Aflatoxin Challenge: Round 2 with Insertions!( Posted by ianh 82 2278 | Tue, 10/31/2017 - 17:26 | 0 comments )
Destroying a Silent Killer in the Food System
We are launching an uncommon collaboration between UC Davis, University of Washington, Northeastern University, Mars Incorporated, Thermo Fisher Scientific, and the global public, to engineer a protein tailor-made for the degradation of aflatoxins.
Imagine a naturally occurring poison – known to cause liver cancer – which 4.5 billion people around the world are chronically exposed to1.
You don’t have to imagine. The poison exists in the form of aflatoxins, which are compounds produced by certain fungi that can grow in or on almost all grains and groundnuts1. Aflatoxins are known hepatocarcinogens, meaning they cause liver cancer, and are considered a Class 1 carcinogen by the International Agency for Research on Cancer, part of the UN World Health Organization. Liver cancer is the third leading cause of cancer death globally, with 83% of cases occurring in East Asia and sub-Saharan Africa2-3.
Beyond liver cancer, aflatoxins are associated with worsened health outcomes in the developing world. These fungal toxins have been shown to negatively impact the immune system, rendering vaccines less effective, and to reduce vitamin absorption and recovery rates from malnutrition4.
Aflatoxin in Our Food System
So how does this potent carcinogen get into – and stay in – our food? In developed countries, laws limit the amount of aflatoxins allowed in food for humans, livestock, and pets. These laws are backed by expensive monitoring and advanced food safety technologies.
In the developing world, aflatoxin limits are set but often unenforced. Under-regulated food manufacturing, subsistence agriculture, small-scale farming, and lower healthcare budgets all contribute to the aflatoxin contamination problem. Over the past 50 years, when food and feed has been sampled in developing countries, the majority has been found to contain aflatoxins well above the legal limits5. A recent study in India found that 100% of collected samples of grain flour were contaminated, with an average aflatoxin concentration three times higher than the limit allowed in the United States6-7.
Aflatoxins affect more than just the plants we eat. When fed to cattle, they transform into a more potent toxin which accumulates in milk. A 2016 study in urban low-income areas in Nairobi found that 100% of the milk tested had detectable levels of aflatoxin, with 63% of the samples above the legal limit8.
This global health issue is also a trade and economic problem. Before scientists understood how harmful aflatoxins can be, Africa accounted for more than three quarters of the global peanut export market. Its current share is about 4%, partly due to its inability to meet aflatoxin standards outside of Africa. This costs Africa $1 billion per year in lost peanut revenue alone. Other African crops are similarly affected.
Climate change may make the world’s aflatoxins problem worse. The fungi which produce aflatoxins thrive in hot and humid conditions. Right now many parts of the world are not well suited for these deadly fungi, but rising temperatures could allow for more growth in more areas, increasing the threat of exposure. In just the last five years, cases of aflatoxin contamination have been increasing in Europe9.
Mitigation of Aflatoxin
How can we tackle aflatoxin? Removing aflatoxins from our food is extremely difficult. To date, there appears to be no ‘best practice’ for minimizing exposure other than strong enforcement of legal limits and systemic use of safe agricultural practices commonly adhered to in developed countries.
Several approaches for managing and degrading aflatoxins are currently in practice, but none are widely considered effective. At present, microbial, enzymatic, and bio-control approaches are preferred. Each has its own complications, ranging from expensive multi-day treatments to low efficacy in certain environments. The underlying issue is that there is no cost-effective and efficient system to remove aflatoxins from our food system5,10.
Our Challenge For You
Through Foldit (run from University of Washington and Northeastern University), anyone in the world can help to optimize an enzyme that we hypothesize could be capable of degrading a susceptible lactone ring in aflatoxin. Chemical degradation of this lactone ring has been demonstrated to decrease aflatoxin toxicity by more than 20-fold11. However, the enzyme in its current state is unable to perform this reaction. We are asking Foldit players to restructure the active site of this enzyme.
The goal is to enable the enzyme to effectively interact with the partially degraded Aflatoxin B1 molecule that we have modeled inside. Once we develop a highly effective enzyme capable of degrading the toxin, enzymes could readily be added to feed and food to remove toxins in real-time, complementing the current techniques for aflatoxin management and control12.
The Siegel Lab at the UC Davis, through the support of Mars, will select the top player designs to experimentally characterize. Thermo Fisher Scientific will donate gene synthesis services, encoding player designs through its proprietary DNA synthesis platforms. Using these genes, researchers in the Siegel Lab will produce the player-designed proteins and determine whether they are capable of degrading Aflatoxin B1, the type of aflatoxin that is most potently carcinogenic.
Over the next few months, we’ll be releasing a series of puzzles in which Foldit players can manipulate the enzyme model with escalating degrees of control (starting with simple site directed mutagenesis and leading to full redesign with insertion/deletion of protein segments). We will continue to provide feedback on previous rounds of player designs in order to further explore this enzyme’s ability to degrade aflatoxin. The first puzzle of the series, 1440: Aflatoxin Challenge: Round 1, is available now!
By participating in these Aflatoxin Challenge puzzles, the players agree that all player designs will be available permanently in the public domain, and the players will not seek intellectual property protection over the designs created as part of the challenge.
1. Williams, J. H.; Phillips, T. D.; Jolly, P. E.; Stiles, J. K.; Jolly, C. M.; Aggarwal, D., Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004, 80 (5), 1106-22.
2. Schatzmayr, G.; Streit, E., Global occurrence of mycotoxins in the food and feed chain: facts and figures. World Mycotoxin Journal 2013, 6 (3), 213-222.
3. Ajani, J.; Chakravarthy, D. V. S.; Tanuja, P.; Pasha, K. V., Aflatoxins - A Review. Indian Journal of Advances in Chemical Science 2014, 3, 49-60.
4. Liu, Y.; Wu, F., Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ Health Perspect 2010, 118 (6), 818-24.
5. Williams, J. H.; Phillips, T. D.; Jolly, P. E.; Stiles, J. K.; Jolly, C. M.; Aggarwal, D., Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004, 80 (5), 1106-22.
6. Kumar, P.; Mahato, D. K.; Kamle, M.; Mohanta, T. K.; Kang, S. G., Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management. Front Microbiol 2016, 7, 2170.
7. Ramesh, J.; Sarathchandra, G.; Sureshkumar, V., Survey of market samples of food grains and grain flour for Aflatoxin B1 contamination. Int. J. Curr. Microbiol. Appl. Sci 2013, 2 (5), 184-188.
8. USDA, Mycotoxin Handbook. 2015.
9. Kiarie, G.; Dominguez-Salas, P.; Kang’ethe, S.; Grace, D.; Lindahl, J., Aflatoxin exposure among young children in urban low-income areas of Nairobi and association with child growth. African Journal of Food, Agriculture, Nutrition and Development 2016, 16 (3), 10967-10990.
10. Herrera, M.; Anadón, R.; Iqbal, S. Z.; Bailly, J. D.; Ariño, A., Climate Change and Food Safety. In Food Safety: Basic Concepts, Recent Issues, and Future Challenges, Selamat, J.; Iqbal, S. Z., Eds. Springer International Publishing: Cham, 2016; pp 149-160.
11. Ehrlich, K. C.; Moore, G. G.; Mellon, J. E.; Bhatnagar, D., Challenges facing the biological control strategy for eliminating aflatoxin contamination. World Mycotoxin Journal 2015, 8 (2), 225-233.
12. Lee, L. S.; Dunn, J. J.; DeLucca, A. J.; Ciegler, A., Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity. Experientia 1981, 37 (1), 16-17.
13. Olempska-Beer, Z. S.; Merker, R. I.; Ditto, M. D.; DiNovi, M. J., Food-processing enzymes from recombinant microorganisms—a review. Regulatory Toxicology and Pharmacology 2006, 45 (2), 144-158.