 I'll thank you very much. I'd like to thank all the organizers for this opportunity to speak at such a great webinar series. Just want to make sure everyone can hear me. Yes, I've been at NIST 10 years. I'm going to talk about some of the modeling efforts for monoclonal antibodies and high concentration formulations. I don't know if I can get this slide to change. There we go. So just really quickly, when I mentioned any commercial or trade names in this talk, it does not imply endorsement by NIST. So there were a lot of scientists involved in various parts of this work I'm going to talk about today. So I'd like to start by just acknowledging a number of people, both at NIST and IBBR and three joint postdocs that we've had with IBBR over the years, who have done some collaborations with NCNR and Lynxis and CNRS. I'm going to present two case studies with pharmaceutical antibodies done in collaboration, one with AstraZeneca, as well as the Stevens Institute. This is the beautiful map included with the Lynxis NIST webinar and Kale did an amazing job with this. So just reminding everyone we're stuck maybe fortunately or unfortunately in the pits of course graining today. So this is the outline of the talk. First I will motivate some reasons why we need multiscale antibody simulations to address challenges in the pharmaceutical industry. And after introducing a few examples of the kinds of experimental data that we're hoping to compare with to validate our models, I'll introduce a few different kinds of models, both isotropic and isotropic models with flexibility in the hinge. And then I'll present these two case studies with real world pharmaceuticals trying to help rank order them using these course graining models. And if I have time at the end, maybe five minutes I'll briefly talk about the software that was used during all of this. That's available to the community. So what is an antibody and why do we care about them. So, on the left here is a molecular dynamic simulation of the NIST mad by Christina Barganzo that you may have seen earlier in this series, and in case you missed some of the previous talks. So it's that antibodies have these three domains that are connected by this relatively flexible hinge region, and they have this characteristic Y shape, where the arms are what we call the fabs at the top here have variable regions that bind to antigens. And the bottom is the, what we often call the FC is more conserved than the fabs. Antibodies are in about, if you look at like pharmaceuticals just in general and rank them by sales you'll see that they make up almost half of the top pharmaceutical products here's a couple of examples. And they're used to treat things like cancer arthritis psoriasis and asthma. So they're very important in industry, and the very large complex molecules we'd like to be able to understand them better and simulate them and help with, with screening pharmaceutical candidates. So, they're big molecules so one of their problems is that they suffer from physical, some physical stability issues so here's an example of viscosity of two different maps as a function of their concentration. And you see as you increase their concentration, the viscosity can go above this subcase injection limit. And that means that they then have to be delivered to patients by IV, which is costly and time consuming so we'd like to be able to predict. So, between map one and map two, we could have predicted this without having to make them first, which is really expensive or make large quantities of it to do the experiments. And also the companies don't really want to share exactly what map one and map two are these are often like highly guarded sort of secret secrets and it's not just the viscosity at high concentration, we also have to think about issues with liquid liquid face aggregation, as well. So, if we want to study antibodies at high concentration and study their interactions with each other, at minimum we have to simulate at least two, which is already 40,000 atoms. And if you want to look at phase behavior and viscosity, that's collective behavior of our long link scale so that would actually require something like a 10 million atom simulation, which is simply not feasible with the standard molecular dynamics kind of approach. We need to develop multi scale and coarse grain models that capture the essential physics of the antibodies, but they have less interaction sites so we don't look at every, we don't have every single atom interacting. We need some kind of more coarse model than that but still captures the flexibility of the hands and specific patchy interactions on the surfaces. The coarse grain models also need to be able to capture the effects of the solution, for example, how they behave as you change the salt concentration, or the pH conditions, as well as if you add the crowders or excipients and the solution. And these are all things that we're, we're working toward. So now I'll show some examples of the kinds of experimental data that we'd like to look at to validate our models. And one thing you're going to hear a lot about in this talk is the second osmotic Vera coefficients. So a quick vignette on these. This is a, the formula for the second Vera coefficient of an isotropically interacting particle, where you have R is the, as the interaction is a function of separation distance. So you can see that it's essentially an orientationally averaged metric for the interaction between two particles. And it's a very, it's very readily calculated in theory simulation as well as measured in experiment. So it's a very nice direct comparison between the simulations and theory and experiment. So what we're going to use in this relatively famous. Noron Frankel, it's corresponded extended corresponding states to collapse phase diagrams. It's very efficiently calculated with mayor sampling Monte Carlo, which integrates these mayor functions in a efficient way. And so we will be using this for the, the models. We also recently published experimental data on the NIST map at low concentration, we have examples of charge as a function of pH for both the full map and the different fc and fab domains that was measured by electrophoretic light scattering. We have a second automatic beer coefficients that were also measured with light scattering, both for the fab arms as well as the whole map, and also how they, we have these values over a range of ionic strengths, as well as pH is, which is a is a great resource for trying to build models that hopefully capture these, these kind of solution effects. At finite concentrations, some of the most informative experimental measurements are small angle scattering. So here is the scattering intensity for the NIST map at using x rays from two milligrams from a liter to 193. And so we'd like to use computer simulations to be able to calculate these compute these, these skirt, these scattering curves, and interpret these interesting features like we would call these interaction peaks. And we can also get effective structure factors from these by taking one of these scattering curves and dividing by the, the, the high dilution curve and getting an effective structure factor, which we can interpret these peaks and fit to them to models, as well as we can readily calculate these with our course grain models and compare. All right, so first I'm going to introduce these isotropic models that capture flexibility and the maximum packing of antibodies. So remember that antibodies have these three domains that are flexibly connected by this hinge. And so one of the simplest models that we could think of for modeling antibody would be besides just modeling it as an entire sphere is modeling each domain as a sphere and connecting them with a flexible hinge region. And so we could improve this model by by modeling each domain as a collection of spheres. For example, here, one of the domains is modeled by two spheres instead of one. And we can see that if we look at the scattering at infinite dilution that's the black curve here for the experiment. The red one is the seven bead and the blue is the four bead model, and you see improvement as you increase the number of spheres to model the antibody, you see better comparison with experiment. But that comes at a cop at a at a big cost that it, it adds, you know, the number of interaction sites means is twice as slow, as well as more complicated bonding potentials and things like that. So look using these kind of models, though is is informative we can see that and and I think I mentioned I should have mentioned again that these are mostly hard sphere like excluded volume interaction models. So we're mainly focused on high concentration so 175 this is pretty high concentration for these antibodies. And we see for the scattering intensity, when the domains are held as far apart as possible. That would be like this red curve here and when and the blue curve is when the domains are as close as possible. And so we see very different scouting intensities we see that the intensity increases, as the domains are closer together. And that's because of the improved molecular packing, so they have a smaller sort of effective size. And you can see that the position of these interaction peaks also changes. So, for the more open structure to the more close structure, you see that the peak shifts to lower q values. And this also suggests that the, the more open structures are interdigitating with each other between. So basically another domain of a different antibody could maybe try and fit in between here. And we're seeing similar differences for the, the higher resolution seven B model as well. We can also look at the the effect of making these models flexible so this is again the scattering intensity of the the solid lines are the flexible fully flexible models and the dashed lines are the rigid models. And we see the scattering intensity is higher. Again, because the flexible models are able to to pack better, and that increases the scattering intensity and have a smaller effective size. So these were some of the first models that looked at flexibility in in the hinge whereas some of the previous course we models just assumed the antibodies were rigid. However, we didn't include anything more than exclusive volume interaction so we're not able to capture effects of the solution like like salt concentration or pH, or specific patching interactions that that may lead to some interesting clustering behavior that are experimental collaborators have observed. So we're looking to to really improve on these models. So that is the anstropic models that I'm going to show you today. Just to briefly review the some of the course grain models that have have already existed literature. This is the one I just described. You can see that there's a lot of different course grain antibody models in the in the literature. And these are, these are really great models. They've been used to compare with experiments and they've been hand tuned for particular antibodies. They take really a lot of effort from a talented scientists to to parametrize these, because you have a lot of these questions like, where do you put the beads how big are they how do they interact right. And so, one of the issues that we ran into with these with with building on these kind of models is the number of parameters of starts increasing and it makes it harder to screen these. Say if the pharmaceutical company wants to look at how these antibodies behave with like 200 different sequences, and the sequences might only change just a little bit on one of these beads or something. Or it might be you all throughout the protein. They also don't want to share the sequence of 200 different candidates they would like to have a software that we could provide where they could just push a button. And with with less parameter choices and get some results. Right. So, one of the things that I proposed when we first started meeting with the links is simulation working group was going beyond these kind of spherical models, going beyond in the physics community we call these spherical cows. Trying some anisotropic model so here's an example of a model of a monoclonal antibody that I that I showed a few years ago as an example where you could model the fab arms as super quadrics and you can model model the or the fc as super to rights. And this builds on some of my previous work in non antibody applications. But when Sergey shared the work that he was doing and presented last year on this docking based method of simulating proteins I was I was really inspired. So, so briefly, the way I would describe this is, if you treat these two proteins as like a ligand receptor, you can do this very efficient docking algorithm to find the most favorable configurations. And then there's a Monte Carlo move that randomly samples among those those configurations. And this is a really efficient method for simulating hundreds of thousands of proteins that are very crowded where these favorable interactions dominate. Now, in cases where the favorable interactions may not dominate or some of the, the repulsions are just random configurations also play a role. I'm not exactly sure how you would use this method to compute things like second automatic miracle efficiency. Or scattering intensities and cases where there may be less concentrated, or there's repulsions that are dominating. But I was very inspired by the idea and I thought that what if we had represented a protein with an all out of model to begin. And then we looked at decorating the surface with what you can think of as like binding sites so here I'm showing lysis I'm decorate where the surface is decorated with these pink loft beads with a 15 degree resolution and the solid angles. And so I can do sort of an angular scan where each one of these pink beads is a site for looking at how another lysis I might interact. There are many different orientations of the of the particles and if you roll something about the size of a water on the surface, you get for the 15 degree resolution the solid pink surface, and for infinite resolution, you will get the blue. So you can see that a 15 degree resolution the angles is actually not a terrible representation of the of the entire protein. It's a representation of getting the action level at least getting the shape. So what I can do, and what I'm proposing here is that we could take one domain of the antibody and fix it, and then systematically rotate the another domain about it, and calculate those interactions using an all out of model that you store. And then during the course of during the course grain simulation, you can interpolate from that those pre computed interactions. So if you have two rigid bodies there's six degrees of freedom that you search over. If one domain is fixed then the center of the other domain is given by three coordinates and then its orientation is three more so you could think of looking at this in terms of one distance is five angles. So this is a comparison of taking a second all out of approach versus the pre computed in a traffic model. So this all out of model for an antibody domain would have about five and a half thousand atoms or interaction sites, which is feasible for simulating a single protein. If you want to go for the anastrophic approach. This would be a single interaction site that has an anisotropic that has that depends on the orientation of the particles, the relative orientation. So to begin you have this expensive calculation of, of how they interact over 15 degree resolution with five angles which turns out to be over a million orientations. But once that's complete then you have your simulations are about 1000 times faster than the auto model, because it has about the same computational cost of a three site. So the all out of model is as if this had three atoms. And there are some costs involved in this approach though. There's of course you have to store the table could be about 100 megabytes or more, and with with the 15 degree resolution. You also introduce a number of approximations. So the all out of model needs to be assumed to be rigid to make this practical. Otherwise you have to do an ensemble average calculation for every one of the million orientations. And we're also going to make the implicit the solvent implicit, so that we don't have to integrate over the solvent degrees of freedom. And we also assume that we can interpolate between these 15 degrees so if 15 degrees is too big of an angle and you miss some very important patchy interactions, then you're just interpolating between two less favorable configurations and you miss that very important interaction so that's another approximation that we have to consider. So this is the all out of model that that I used in this work. It has excluded volume interactions from amber and auto doc. And it has since there's implicit solvent, it has a screen charge term with this Kappa parameter that is determined by the temperature and, and the ionic strength or the concentration of certain chloride. And the charges also signed by a parse and the pH determines the charge states of the residues. So we can capture some effects of pH and the salt concentrations. It also has a Vanderwall short range attraction with a fitting parameter and front here that's the one fitting parameter from from this work that was published. So here's six different, I believe six or seven different proteins over a range of ionic strengths and pH. So here's an example of the second osmotic virco efficient as a function of ionic strength where black is the Paul Adam model, and the various color symbols are from various experiments. So here's an example of a potential energy curve for two fab domain of the arms of the antibodies that are fixed at just a arbitrarily chosen orientation. And so I'm looking at their, their interaction energy is a function of distance between the centers. So a little less than 60 angstroms is where they overlap so below that point, it would be infinite, exclusive volume potential. And you can see in this case for this salt concentration. There's this short range attraction that goes to basically zero toward a cutoff. And, but if you decrease the amount of salt in the solution, and you, you look at ionic strength of 50 millimolar instead, you see that the the charge interactions aren't screened as much. So now you have this longer range repulsion that's above zero here, but as well as a short range attraction which is an important feature of a lot of these kind of models that you'll see in the in the literature that and this model also captures that. But it also depends on orientation and things like that. So one of the major assumptions that we make in this approach is that the structure is rigid. So here I'm showing very coefficients with ionic strength for a number of different PDB structures of the lysis I'm protein. So these lines here these four different ones are four different PDB structures of lysis I'm. The color symbols here are various experimental data. So here I'm pointing out, you know the effect of this rigid assumption in a way is tested by looking at different configurations and we see differences in the very coefficient, roughly on the order of the variations that we see in this model. I also included this all atom curve here which is it so these were using the coarse grain 15 degree resolution model, whereas this purple curve is the all atom model, which doesn't have the the resolution issue. This is done with a mayor sampling. So, so it's interesting to see that the all item was actually sort of over predicting the viewer coefficient, but through some fortuitous cancellation of errors, the course grain model actually matches the experiment maybe a little closer and some ranges of ionic strength at least. As you get to higher on extreme. Your, your screening the charges more and so an experiment it goes down more than you, but in the course grain model you see it hits this plateau, and that could possibly be because you're of the interpolation assumption where you're missing some very important. The most attractive interactions might be missing in the model, and those contribute very significantly as you increase the salt. So this is only valid in certain ranges of ionic strength you have to carefully consider when you're doing the course training. So when I showed this to Michael and Sergey about a year ago. Michael proposed to look at this lactoferrin protein that he's looked at in the past that has this very interesting patchy interaction at intermediate values, intermediate salt concentrations. If you decrease or increase the salt, then, then the repulsions increase so you have this interesting minima and he's sort of challenged me to see if this course grading approach could capture that. So, these orange points are the experimental second osmotic very coefficients, and the red are the all Adam and the black plus symbols are the course grain model with an 18 degree resolution. So you see that the auto model does capture this minima in the osmotic vehicle efficient. And for the course grain model, it does have a minimum but it's not at the right position and that again is likely because I use this 18 degree resolution to model and it may have missed some of the most important patchy interactions. And this is a very big protein this is much bigger than the license I'm. In terms of art angles. This is actually very big spacing between the various orientations that we're looking at with 18 degrees, right. So we could improve that instead of modeling this lactoferrin as a single one single site, we could break it down into multiple sites, for example, and we could even to do something like a flexible bond between those. And even if we did for these smaller collections of the atoms that we assume are rigid. Even using the same resolution, the arc length is now smaller so the, we could see some improvements there. And also I'm just introducing this idea of breaking it down into multiple sites, because that's what we essentially had to do with the maps. And I talked a lot about this is showing a course grain antibody simulation of a single map, and I talked a lot about the interactions between these domains, but how do we model the flexibility of this this highly flexible hinge and link a region. And the way that we do that in the course grain simulations were used confessional bias Monte Carlo to randomly select from the angles and links here in the in the link in the hinge region. But those were informed by all Adam molecular dynamic simulations done by Christina. And let me show an example of that so here we have probability of angles which are for the solid line these are from the all Adam simulations. And they're the angles of these elbows here essentially between. So the black line would be the FC angle from the FC to this elbow to the center of the hinge. And in the course grain model we're going to just assume all these angles are are the same. For the solid line. So the solid lines I'm shown here are from the all Adam simulations which include multiple ionic strengths. And I'm, I'm pointing that out because there's, there could be some very highly specific interactions between these domains that influence this probability of these angles that you're seeing here. But the most important thing that we really wanted to get from the all Adam is what is this minimum angle that we can that we're likely to observe from an all Adam simulation. Obviously, 180 is the maximum when it's straight. And so the what I'm showing here for this course grain curve with the dashed line is having only excluded volume interactions between the domains, when I'm not including the, the specific interactions between the domains those will be included in the simulation, but for getting this this power ability curve. This is essentially like a sign distribution you would expect from randomly picking angles but they were cut off at a certain minimum angle. So the autumn informed this, this minimum angle choice. And we did the same sort of approach for the bond links as well so that's the bond links between the center of this hinge and each of these these red points where it connects to the rigid domain. And so, from the all Adam we get the minimum and maximum bond links and without with only excluded volume interaction the course grain model we see distributions like this. And this is a this is a big improvement on on previous models that either that often would get this information just from like a single crystal structure or something like that. That is the model that we're going to use for these two case studies that I'm going to show with some, some real pharmaceutical antibodies. So, again, this this idea of from from the introductory material. Maps can have very different viscosities. And that can be something that's really important in to the companies that they would like to be able to predict early without having to make a bunch which is really expensive right. The companies have come to us and we can't share too much information on the specifics of the antibodies so we're just going to call them a B and C, but if we did this case study in collaboration with the companies where we have been that they give us this the sequence of a B and C. Are we able to predict which ones are going to be problematic and development. And so, just with given the sequences, we're able to get the structures by homology modeling from the NIST map structure by Christina Burgonzo. And then we apply the course grain methodology I just described to these, these structures, and see if we can predict the which ones would be problematic and which ones were not. So, here is a sort of top down view of the fab arms of each of the candidates A, B and C, and the atoms are color coded by the type of residue if it's hydrophobic or positively or negatively charged. And if I had audience participation I might ask someone, you know if they could tell me which one of these three A, B or C, or to which ones look like they could be problematic or which ones might have high viscosity. You know, maybe someone much smarter than me could could figure this out. But I, I'm going to try and just use a computer to apply this this kind of push button method without any adjustable parameters and see, see what happens. So, here are example simulations of map A and B using the course grain simulations these would be these are 50 grams per liter, six pH, 150 millimolar salt. And so these would be a million atom simulations, if we were using the just a standard molecular dynamics approach. In the course grain model I could run this for a few hours on on a laptop this laptop I'm presenting and, and get reasonable results. So, I would say that I can't really tell much of a difference between map A and B here just by looking at it but we can compute properties the probability of the fab arms coming into contact for example. So, from the center of mass of one arm to the other is the distance that I'm showing here. So around, if you remember for around 60 angstroms is is roughly the contact distance. So the showing the pair distribution. So the map B has a much more likely probability of the fab arms coming into contact than maps A and C. And so, we know that's problematic based on some of the previous course grain simulations this was figured by Marco a few years ago that we find really useful so let me walk through it. The second very coefficient against a lower very coefficient means more traction. And on this axis, the x axis has the basically the ratio of the attractions of the FC versus the fab. So if the FC is more attractive than the fab you get these cases where they form these micellar like structures where when you when you have enough of them, essentially the fab arms kind of crowd out the possibility of adding more. So that they make these finite size clusters, whereas if the, if the fab arms are the more attractive ones, then the antibodies can essentially, there's two arms to these antibodies right so they can hold hands and and form these transit clusters that can span long distances. And if the FC and fab are equally attractive, then you can get things like liquid liquid face separation and anywhere else in there, we would hope would be like a stable solution right. So, these candidates all had the same FC, and they had different fab spoke well if map B has a much higher fab attraction, then it seems that it's more likely in this transient cluster region than the other ones. We can also look at the concentration dependence of these distributions so here we're going from 50 to 200 grams per liter, and we see that map B has relatively concentration independent peak so that kind of tells me that map B is finding the other fabs, regardless of the concentration, whereas A and C are sort of as the concentration increases they're just so happening to finally find each other. We also can look at the vir coefficients of these antibodies, we see that map B again has a low vir coefficient which means more attractions, and a and C seem to be very similar, and much higher. So the are our collaborators were happy to see these results they say. Yeah, this is pretty much what we knew about these antibodies before we gave you gave you the sequences so, so that was a successful first case study so now on to the second one. In collaboration with and quant law. So we looked at in the past 27 different antibodies and this is showing their viscosities at 150 grams per liter for these 27 in this work for within the chat group as well. They use machine learning to try and predict these these viscosities. pink one shared the structures of four different antibodies from this group, including the highest viscosity ones maps 17 and four, as well as low viscosity 10 and 27. And so we're going to apply this course grain methodology I described and let's see if we can make some predictions. So this is showing the vehicle vision again lower vehicle efficient has higher traction. So Mab 17 had the lowest vehicle efficient which is also the highest viscosity, and for had the second highest viscosity and the second lowest coefficient. So these are pretty promising results for this course grain approach maps 26 and 10 are the ones that have the lower viscosity, and they have the higher vehicle efficient so just using the vehicle efficient we've already sort of been able to rank order these in terms of their viscosity by core just correlating them. So, if we look at the fab fab interactions. This is at 150, 150 salt 100 grams per liter. We see, again, the, the interactions between the fabs are highest for the green map 17 which had the highest viscosity. And, and the red was low right, but there's this interesting case here, what happened to have four and and 10. So 10 had a low viscosity and for had a high viscosity but their fab interactions seem to be about the same so so what's going on here what are we missing. So I'm going to go back to this amazing pot that Marco made years ago. And so, in the previous case I kind of glossed over that all the fcs were the same. Well in this case study the fcs are not the same. So we're missing this information we need to know. We've looked at the fab fab interaction but what about the fcfc interaction okay that's going to help us distinguish between the blue and the orange one right the blue had the high viscosity. Okay, so we look at the fcfc interaction, blue is the blue one is down here with red, but the orange one is almost as high as the 17 that the high viscosity. So, what that means is that the orange one is more in the center of this and this axis, because both the fc and the fab were high interaction. So it would be somewhere over here, but in the in the blue map for case, since the fc interaction was was lower it pushed it into this client transient cluster regime. So, to conclude the first part of this talk. I would like to say that, given just the map sequence, and some autumn force field taken from the literature, we're able to use this course learning approach without any adjustable parameters to make accurate predictions of the high concentration behavior of the antibodies that were using these pre computed course grain models. So, this approach is actually useful for a lot of complex models not just antibodies, even, and it's a code that I made open source and available to the community. So if you'd like to use it. You can send me an email herald.hatch at this dot gov or visit the software website feast with two s's. And yeah, I'd be interested in using this this methodology and helping the community. So, I'm going to take about five more minutes really quickly to talk about the software that was used and all of the results I just showed you this in this talk. Okay, so why do I even care about software. Well, I've seen firsthand how something as simple as a coding error in what happened to be a sophisticated Monte Carlo technique that wasn't available open source could lead to a seven year dispute. And finally be solved when the, when, when the codes were made available. And this also has a very important role to play for standards in software so so simulations, even play a very important role in, in, in scientific method, because when you compare simulation experiment, you get a handle on the model approximations that were made. So in simulation theory you get a handle on the theoretical approximations. And just as NIST has an important role to play for standards in these experiments NIST also has a role to play for standards and simulations. So in our group. This has been available for I even joined the group we have this NIST standard reference simulation website that I used as a, as a young graduate student, even that provides a lot of well tested data. These are simple models that you can use to to test your codes. Also, why do I make the software available. Well, there's a need if you look at the community there's a need for Monte Carlo software, because if you rank order scientific software by citations. You see there's a lot of well used quantum and molecular dynamics codes, but you have to go down toward the bottom to find the first Monte Carlo rasp is an amazing code. I'd like to see Monte Carlo just use more generally in the community, because it has a lot of methodology available to it. Like mayor sampling gives ensemble five histogram and Grand County go ensemble simulations. So these are made available in the feast code that I've developed and, and it has two S's because I wish it was as cool as Lance. It has the, the anthropic tabulated potentials that I talked about as well as some of the cool conversion bias stuff. I'm not going to go through this whole list I'll just show a few videos in the last couple minutes of the talk. So, here's an example of flat histogram Grand County called Monte Carlo simulations, where the volume fraction is changing this red dollars corresponding with this instantaneous configuration of the new johns particles that are being inserted and deleted from the and we can get, we can look at the, the power ability of transitioning between different numbers of particles, and that allows us to calculate the probability of the vapor and the liquid and rigorously calculate face diagrams that way. Also showing examples of circles that are slowly morphing into squares by random Monte Carlo trials and shape expanded by histogram simulations that we can use to look at how the shape changes the kind of stable structures that would form. And this example had cylindrical colloids that had an attractive interaction due to deplete in the solution, and they, they gel and we had some comparisons with experiment there. We also have cases where, for example, the course brain antibodies might form clusters like these. And so you need rigid cluster moves and Monte Carlo to be able to efficiently sample these and those are available in the code as well as conventional bias aggregation volume bias methods to find a favorable relative orientations between flexible molecules. So to wrap up the talk again I'd like to just acknowledge all the many people and institutions involved in this project, and thank everyone for their time I'd be happy to take any questions.