 very nice to be invited to come here and to participate in the organization, although I have to admit that actually Tommy's done most of it and OSA. So thank you to both for that and I have not really had nearly as much to do, so I'm very grateful. Also, I have to start off with an apology actually and I apologize for not being somebody called Mikael Hartlein, because originally when we planned this presentation we wanted to get an expert in molecular biology and in my group, Mikael Hartlein is that person, he is an outstanding molecular biologist and he has much more insight into the molecular biology and the biochemistry of what we've been doing for the in my group. I'm actually a biophysicist and so I work, if you like, on the other side of things and but as Tommy said, I've been integrally involved in developing this, proposing this facility and in developing it over well, nearly 20 years it's been going and I think as a result, you know, lots of other facilities, including in London have started to put together facilities like this to support biology, but in the end, if you don't use deuteration and you don't to make your samples to optimize your samples, you very much narrow the gap between what you can do with x-rays and what you can do with neutrons. So without that, you know, you're not an awful lot ahead of or different from what you can do with an x-ray source and of course x-ray sources have huge advantages in their own right or intensity and bean size and all sorts of things. It was in the early 2000s really when I proposed to build up a facility of this type for neutrons and it actually met with quite a lot of opposition to be set up and it was all very complicated. So I hope, so what I'm not going to do is to tell you the molecular biology details of every deuteration method that we've developed and how they've been applied. I'm going to point out more on the application side of deuteration and that will relate to different regimes for deuteration. So I'm not going to get into the molecular biology that it'll become pretty obvious what organisms we use for expression and some of the techniques are quite complex, but you'll get a rough idea of what's the something we do and the way in which it applies, how can we apply it to neutrons scattering of different types and I'm aware of the fact that I may repeat, I will try and, I didn't watch all of Frank's lecture this morning and I know ESCO will have spoken already, is that true? And I may over that accidentally a bit, so I apologize if that happens, but I'm trying not to go over stuff that I'm almost certain they will have told you. So it'll be about what we do and some examples. So anyway, please yell if you want to ask a question or yell at Tommy or somebody who I don't mind stopping, we can talk for as long as we want. So this is our site, many of you I guess will have been to it or will go to it at some time in the future. Obviously, ESS is being built and will become functional but until then, this is one of the best sources around if not the best source and that is the reactor that you see on the left of your screen here. It's a European infrastructure is also an infrastructure to which Sweden subscribes and as such it is entitled to apply for being time there. And so it's something that you will either do or should be using or can use and it's adjacent to that of course is the synchrotron and the synchrotron was not put there just because there was space. It was put there very, very specifically to co-locate so that the complementarity between the two capabilities the x-rays and the neutrons could be fully exploited. And also the restaurant was not co-located by accident either. But anyway, so in between the, those there is the EMBL outstation which you'll see here. So I've actually got labels for these, there's the ILL and here's the ESRF and the IBS is off this picture unfortunately I think they should retake it and get at the IBS in there as well and then EMBL outstation. So EMBL outstation was actually built or installed as a relatively small thing in the early days of the ILL, ILL's operations to support biology at the neutron source before the synchrotron even existed. So, but it's been an important thing because without that it's very difficult to do biology if you're not in close proximity to good lab capabilities and that's even more so true now than it was then. I mean now there's all sorts of things you do with biology and biological systems that you couldn't do then when EMBL was first located there. And then of course the synchrotron came along and the EMBL became closely connected to that. So EMBLs had a strong role in the biology on this site since its inception since the ILL existed. And so there we are, and then we have formed partnerships. Now the partnerships actually occurred because despite the fact that these things were co-located, it was very easy for the neutron people and the X-ray people to stay in their own corners and not to interact in the way that was officially hoped at the beginning. And that was why the partnership for structural biology was built. That was built, it was formed. The partnership was formed in the early 2000s and it has grown, so it's the oldest partnership and it's become very, very strong, a very good partnership. And it was designed so that everybody could share their own platforms, their own capabilities to optimize the science so that we were using more combined techniques and we were pushing more into disciplinarity and multi-technique approaches. And that has been extremely successful. That's the PSB, the symbolic center is right here in the circle. It's the main thing I'm going to talk about which is why I made it green. And it is a bolted on, if you like, to the EMBL outstation, so you can just walk from one to the other. And it contains mixed labs from ILL, ESRF and IBS and so on. And it has a very, very good atmosphere and lots of students, young people, postdocs and so on, who are interacting on a daily basis, very, very nice atmosphere. Of course, that's the sort of thing that's being foreseen now, I think, for Lund. This is the closest I could come to, which is a very visually nice picture, but showing infrastructure developments and the ESS of the future, Max4 and so on. And the base in between, which will be important for forming a scientific cult and enabling a nice scientific culture and capabilities for the future. So, back to this, if we don't think back to zero, and I apologize if I'm going too far back to zero, just to give you a sort of a visual grasp of why we have these two capabilities. And again, I apologize if it's too simplistic, but at the synchrotron on the right, you've got, you've got X-rays, so you've got X-rays and X-rays are very good at seeing stuff where, you know, where you've got large numbers of electrons, because obviously the X-rays are scattered among the electrons, so you have more electrons, then you're going to have more visibility. And the heavier your atoms, the more the stronger the images are. And of course that immediately brings to mind the fact that you have a fundamental problem there because the element with the least number of electrons, which is hydrogen, has very little visibility at all, even in high resolution studies. So you have a problem right from the beginning in that you have hydrogen, which is one of the most important atoms in biology for reasons that I won't even need to tell you, hydrogen bonding and specificity interactions and ligand binding and all sorts of things. And you have a very poor visibility and that's a problem in a lot of cases. And this is the story for the neutron equivalent of the same molecule, in fact, and you can see the difference, right, so you see just looking at this left image, you see the hydrogens are all there at very clearly visible, you can see a hydroxyl group here, you can see the orientation of that hydroxyl group, you have no idea what it is in the X-ray case and so on. So that's fundamentally in a nutshell, you've got hydrogen, you've got visibility of hydrogen that as deuterium, that is much stronger than you can get any other way. You can get crystallographic information, for example, not just this log, we were going to talk about other things as well. And you can get this type of information in exactly the analogous way that you do with X-rays, but there are a number of important consequences of using neutrons and most of that comes down to samples. So you can build the best instrumentation in the world and RLL has got the best instrumentation in the world, but if you don't optimize your samples, you're not going to make the best science, whatever you do, however much instrumentation you throw at it. So anyway, and here's more, more, more, more, more water. Look at, you can see your H's as well as the oxygen in the middle, you can see the hydrogen, this is high resolution neutron crystallography, and you can just see these amazing water molecules, which of course in X-ray crystallography you just see as opposite to this really. So you get all that information, orientation of water molecules, you get information on protonation states or amino acid resins, you get all sorts of details that stuff that lies underneath the surface of X-ray crystallography in it, you just don't see. So very often what we find, if I just take the crystallographic angle as an example, very often what we find is that even when you're looking at something and you don't really, you think for example, and I give you an example of this, you find things that you're just not expecting, you're not even part of what your proposal aimed to find, you just find stuff that's appearing because you can suddenly see new things and that's, there's been a lot of that in neutron biology work over the years. And this is an example actually, this is rubrodox, which is a redox protein, which shifts electrons around, if it's a redox protein, it's dealing with electrons and obviously you're dealing with electrons, you're also dealing with protons because, you know, absence of charge and so on. So it's shunting charge around. So, but the reason we actually did this study was I said to a postdoc, I said what would be really good would be to have a standard sample, a standard biological sample, what would be a nice easy one to do and we ended up deciding on rubrodoxin because it's a nice standard system and you can grow large crystals so my feeling was just have a standard biological sample, we put it on the de-factometry, take it on, take it off for all sorts of good reasons that would help us on the instrument and so on, on the de-factometer. And, but then when we actually looked at it, you can see what we're finding, we're finding here, for example, top right, that's a hydronium ion, right, so you couldn't see that with x-rays, you couldn't distinguish a water, a hydronium or even, you know, an OH, so you can't actually distinguish between them. So what we were finding is hydronium, which of course is charged, and what we found was a very, very detailed, here's water, right, very clear orientation of the water, and what we found were networks of hydronium and water that actually formed complex chemical ions that I never even thought about in my life called zondolions, which are complex, so you can see them down here, that's a zondoline, right, it's a complex, you know, complex, it's a complex ion, if you like. So that's just to show that sometimes, well, it reminds us all that science, there's the stuff that you know you don't know, and then there's the stuff that you don't know, you don't know, and sometimes that can be the most interesting of all. So that's just one example, x-rays, neutrons, looking at them together, and this of course is, this is called a visual representation of the Grotus mechanism, showing how conductivity, trying to explain the conductivity of water, as well as the shunting protons across tunneling, I mean, charged tunneling in a water network, and it was proposed, as I said, as an explanation in about 1800 for the conductivity of water, and that's from this type of study is exactly the sort of thing it makes you think of. Anyway, so there we go, a few examples there, this is a partnership building, this is, of course it has the usual mandatory right-handed elix as a fire escape, which is almost reassuring, and these are, we have lots and lots of PhD students all interacting, and a lot of formal and informal activities and so on. It's a very fertile environment, and I imagine that's what everybody's hoping will build up as the ESS Max4 developments and the village in between them starts to grow. Yeah, so, of course, what we're doing these days is, we're trying to build, the most interesting action is actually, whether you look at it in terms of disciplines or length scales, it's in the gaps, it's the bits in between, the bits in between biology and physics, the bits in between, you know, the length scales, I mean, if you look at it, if you think of it, if you stand back a bit, then you think that, you know, we're quite good at molecular things we can do, because we can do solutions, we can do all sorts of things, at a molecular level, and we get lots of, we're not enough, but we get lots of stuff going on there, and then at the other end, we've got organism, organisms, tissues, but quite good there, we can play microscopy and all sorts of different microscopies. And then, but then there's a gap between the molecular level length scale and the cellular structural biology and that's a big important gap where lots of things are changing because microscopy is changing a lot. And I think this is where we realize that we, and this is what's happened really in the last sort of five years or more. So there's been a huge shift in emphasis, away from the classical structural biology, which was largely x-rays, until quite recently, and that has now shifted. So, to multi-technical approaches, integrated structural biology, if you like, is the latest phrase and lots of things are going on there all the time. So it's a very, very rapidly moving field now. And all at a time, of course, when the biotechnology, the ability to produce proteins, express them and use organisms to produce molecules is also growing at an amazing rate. So it's a very exciting time. And so, and of course, if you think about it biomedically, another important gap is between the molecular level biosciences where people looking at ligand drug interactions and all sorts of things, and clinical research. There's a big gap there as well. So it's in fundamental biology, but it's also in the gaps between clinical and molecular studies. So, if I come back to us and look at the type of capabilities we have access to on a routine basis, they sort of can be broken up into three areas. There's sample preparation ones on the left, there's characterisation techniques, and then there's the sort of bigger machines, the structure and dynamics capabilities, the neutrons, the X-rays, the high-field NMR, electron microscopy, and so on. And of course, as everybody knows, the electron microscopy has made huge developments over the last three, four, five years and there's been amazing things happening there. But we have to, and that is actually affecting the scope of X-ray methods, I think that's true, because some of the larger systems are now being doing rather routinely at 100K in these machines, and in an area that actually the X-rays were dealing with up until that point. Okay, so the two techniques are sort of overlapping, and I guess everything's shifting around a little bit to accommodate that, but relatively little has, there's been relatively few consequences of that development for neutrons because we still, you know, you still got the same problems, you still got the hydrogen problem, you still got, so that sort of emphasises the fact that the neutrons really are still as relevant as before, even despite this development. So anyway, so amongst these platforms, the ones that are relevant to neutrons are the ones highlighted here in green. So we have the neutron, the deuteration of arteries in my group, which is a life sciences group. So we have that platform, we operate that platform as a user platform, you, any of you can apply to use it, you can write a proposal saying you want this deuterated or that deuterated or something. We tried to, we have an interest in large crystal growth. And, yeah, and on the big machine side of things, we've got obviously neutron, we've got three, four defectometers that can do neutron crystallography. Some of them are Laoy machines, two of them are Laoy machines now, one is monochromatic, and so on. So we've got, and of course we've got sacks and sands, we've got reflection, I haven't put reflection there, but there's a reflectometry, you probably will be hearing a lot about that if you haven't already. So, those are some of the platforms and it's important to realize, I think it's very important to realize that these things develop. You know, it's not as though the biologists say, hey, we've got this problem, we need to have this technique, that's not the way it works. I mean, the initial days of neutron work actually biology wasn't the center stage, it was important but not center stage, and sometimes capabilities emerge because technology has developed. So it's a two-way process, and that's a very important thing to remember in the context of service institutes where these developments are pushed very heavily and effectively by people who run beam lines and who work their feet, who run their feet off and who work their socks off in making these developments. So I think it's important for your future career to know how important those developments are and how much effort goes into that and always to remember it in the background. So anyway, this is the PSP, this is our last external review. There'll be another one shortly, those are the number of publications came out of it. The number that were multi-institute is quite high and the impact factor is all quite high as well. So it's a good collaboration in all these things. And, you know, those pictures are just supposed to highlight the different organisms that are used in producing the different proteins and so on that we do. Now, for degeneration, there's a review I think I sent around that you may have seen, and this is one of the pictures from it, I think we use this. And the review, it was Methods and Enzomology, it was by Mikhail Hardline and the rest of us, or some of us. And we, one of the things that was in it I think was this sort of scheme whereby we highlight different types of labeling that you might want to do. So the blue is supposed to show unlabeled, you could say hydrogenated if you like, but in actual fact strictly speaking, it's natural abundance because even natural abundance, even though sort of there's always a very small fraction of deuterium and in normal life, whatever it is 0.2% or whatever it is. So you can change the blue or to yellow where you completely replace all of the hydrogens in your protein by deuterium. And then we have, we have named a category of deuteration match out deuteration, which is what we call when in solution scattering the overall scattering length density which I'm sure I told you about. So matches that all 100% D2O. Okay, so that's the point at which, if you're talking about contrast variation, you put your protein in there, if it's match out deuterated, it's invisible. You might think well, it's everything to do. But in actual fact, if it's a complex and one bit is visible and the other bit is invisible, then you've got a tool that you can use in all sorts of nice ways. So match out deuterated, produce-rated, I say produce-rated, you know, that's what you want for crystallography typically, for neutron crystallography, because you replace all the hydrogens by deuteriums. What happens then is that you get a massive decrease, as you'll see in a minute, in incoherent scattering, hydrogen incoherent scattering, which gives you a much better signal to noise in the crystallographic data that you can measure. And it has a whole lot of other benefits as well, but basically it gives you this improved visibility, which means that you can get to smaller samples. Of course, samples are the big problem here, because in contrast to X-ray work where you can study a crystal, if it's five microns or less, you need huge crystals. So anything that improves the visibility means that you can use smaller crystals, and that means you can widen the scope of things. So, and then there's all sorts of different things, right? So one of my most interesting is segmental deuteration, where you pick out a particular part of the protein and you label that in some clever way. You can replace all of the particular types of amino acids by deuterium analogues, we've done that as well, that can be useful. And so this is a summary of the regimes that you might want to do to use in terms of the deuteration capabilities that we are involved in. And these, as this is repeating the previous slides on the different expression systems, they think of the different ways of producing these molecules. We do lots and lots of bacterial expression systems, or this is rather standard now. It wasn't when we started, but it's rather standard now yeast, of course, as well. And so it's well free also. And we do peptide synthesis as well. And, and so on. The ones that we are most interested in now in developing are mammalian cell systems, because if you think about especially everybody's increasingly aware of it now with the pandemic, but if you just take examples of, you know, the spike protein of the virus of COVID-19 virus or, or adenovirus or any of these and viruses and then the spikes are the thing that interact with the host. So those interactions are very interesting to study and to know about to fundamentally and biomedically. We call glycosylated protein, so you decorated with particular patterns of sugars, which of course it doesn't really happen. In the case you take a protein you can produce a spike protein bacteria and it won't be glycosylated. And as such, I don't know what that means in terms of the information provided for for the biology. So glycosylation patterns are very important, which is not really something that glycosylation is not something that that's friendly to focus on. It's more a solution thing or maybe the reflection thing or perhaps dynamics. But it is important because it relates to folding, and it's important that you have the if you if you produce it in, for example, insects you get different glycosylation. So you could expect so you actually want to be working in the in the same system or producing your proteins in the same system as the system of interest, biologically. So, but they're much harder to to do trade but we're starting now a program of work on on on trying to develop a made in self deterioration, and I might come back to that later. So anyway, I'm probably going on a little bit too long, you will have seen this picture I assume it's true is it Tommy that this is this picture has been seen from. Yeah, okay, so okay so you know about this right this is a natural contrast. So if we have no juice right so you can see, you know the match out is this is the scattering the intensity of water as you go from 0% to 100 and at various points. You sort of interact you cross the lines for possible liquid or hydrogenate protein and DNA and so on and then up here. I have 100% deuteration and match out labels protein and match up. It as I said before matches out at 100% D2O, whereas fully deuterate protein goes is not matchable. You go. It's too, it's too far gone. It's too deuterated so you can't match it up. But that's, that's not necessarily a problem, but it's just the way it is. And, but anyway, so in terms of no deuteration, if you don't have deuteration then take you can look at a nucleic acid protein complex for example you can. Without any deuteration whatsoever you can make one bit here invisible and the other bit highlights it by choosing different, you know, by choosing that particular contrast. Or you can make another bit go. I go invisible and see the first thing. So you're then able to look at different parts of in this case a nucleic acid protein complex in solution. Which of course is actually, you know, you think about the crystalline world is not biological world it provides huge information in all sorts of different ways but it's not the biological world. And the solution states you might think is more relevant biologically but even that we've got to accept we have an experimental method that we live we work within the constraints of our methods and even the solution state is not perfectly biological because biological systems are much more concentrated than even the most concentrated system we tend to look at. In our experiments and all now, all experiments have their limitations, if it's quite electron microscopy or I think at 100k that's not very natural either. So the relevance of all of these things comes mostly when you put them all together and alongside each other, so that you, you get a collective picture of which you see with different methods and then you say yeah okay I believe it. And so there and then the same sort of arguments for seeing proteins in different violets either in myself, myself or in my cell systems or in, or in, or in planar, planar samples. And I use this, this is something we developed with, actually with Lisa Arliss Cooper and somebody called Selma Merrick who was a student, which she's now at Max Four, she worked jointly between us and Lisa Arliss and Copa Hagen for her PhD. And she developed this thing called stealth nanodisc and you'll see what's meant by that so these are the top right you see there's all these are membrane proteins or cartoons of membrane proteins and the idea is to show how important they are transport, enzymatic activity, signal transduction, neurotransmitters for example, attachment, cellular joining, recognition and so on. So it's a huge amount of importance in membrane proteins and they're also quite difficult to look at because they just are, they're difficult to crystallize and they're difficult to study. There was a development by Sligar a while ago where he developed these things called nanodiscs where he could sort of contain a protein within a small bilayer sandwich, which was wrapped contained by a belt protein around the outside. So that was all sorts of reasons why that was an advantage. But when we looked at that, we thought wouldn't it be wonderful if we could come up with a deuteration regime that would make everything invisible apart from the protein of interest. In other words, if you can make it go like that. And then you could just use standard solutions gathering type methods to look at that protein in its natural context by just using solutions gathering. So that's why Selma call them stealth nanodiscs because they, one part of it just sort of turns invisible and of course there's an airplane called a stealth jet which goes around pretending to be invisible. I'm sure it's not visible anyway. So, so this is what Selma did. She hears the nanodiscs and hasn't got a protein in it but here's the nanodiscs got the belt protein it's got the lipid in the middle. And with the ones that she made you, this is supposed to represent the increasing concentrations of D2O going from left to right so that's finally at 100% they're invisible. And that's where it was published. And since then it's been used and that's the aircraft that I was referring to it's supposed to be invisible as well and that's why she called it up. And there's a few examples here of how it's been used for a transporter and integral membrane protein MSBA embedded in one of these nanodiscs, nanodisc invisible just see the protein and we're doing a lot of this now and actually we're we're hooking hooking up with the electron microscopy people with it with our election we're working with our electron microscopy people to, to put these together the solution state structures and the, and the choir electron microscopy which of course is very much higher resolution but is 100k and you're not going to see transitions going on there and seeing the whole things working together with both methods is infinitely credible. So this is some of the same system with the choir electron microscopy these are the different classes and some of the raw data there at the top and then some of the classes derived from that 2d classes that derive from that. And, and then the structure itself. So, so there's a very interesting sort of move, move there for developing that sort of complementarity, along with the choir electron microscopy. Now, and here's another example so we cholesterol cholesterol is is one of the most important lipids for in biological bio medicine, if you like. And we all know about cholesterol we know about high density of the proteins we know about low density of the proteins we know about the health issues associated with with that. And but the problem has always been for in terms of studying this is that cholesterol as a lipid is basically indistinguishable from all of the other lipids that it is immersed in and functions on site. So you can't actually see it. So what this describes is the development that we made where we took a metabolically engineered strain of Fisher, and to, in order to participate the cholesterol so that we could see it with high contrast alongside all the other lipids in a system. So, this is a sort of a summary of the metabolically engineered East, and that was designed to produce this cholesterol. It was basically the system was was engineered to produce cholesterol instead of a guest or gospel, which is what it normally does. And of course we all know the importance of these things as a hot as a, you know, cholesterol after a score is all these things that was a, there's a huge interest in what cholesterol is doing and not least in some of the vaccines that are being made for delivery to as part of the vaccine program that we all know about in the news. And anyway, so there's some examples there where you can start to get really reliable modeling information on what cholesterol is doing in the systems and, and in the proteins that are embedded in memory so that's a really nice development again, and innovation for a generation we've got a single lesson that comes out of all of this is just that you just have to keep innovating for your samples and that's always been the case for every technique. But it's, it's sometimes the case I think that people just think oh well we can we've got two versions of our trees, that's great, you know, we don't need to invest in them anymore because it's all done. It's a naive argument you to it's never all done, because you're, it's a living subject and things are changing all the time. And it's a bit like when they were sort of saying in the 90s the politicians were saying well physics, it's all done right you know what else is there to learn that you don't need physics departments to you. You can do it, you should focus on the applications and so on. That's also very shallow. So, politicians sometimes get in this situation. So cholesterol become a very important tool and it's producing very exciting stuff. So lots and lots of published stuff has come out of out of this user and using cholesterol and something other people are now producing cholesterol as well in Australia and elsewhere. So, yeah, so those are now right we get back to this log, I started with this one and I'm going to end with an example from crystallography, mostly because it's one I'm very interested in myself. It's slightly selfish but there's the crystallography either blue your protein, it's hydrogenated blue. And, and what you see in the diffraction pattern which is there's a monochromatic perfection pattern just to its right here in the middle, as you see the spots of course from the diffraction but you can also see this dark background. This dark background comes from what I remember Frank, you know, in the early part of his lecture he talked about incoherent scattering hydrogen, incoherent scattering hydrogen has two spin states for those of you who did who remember. There's a lot of ions and bows on stuff that it has to spin states up and down to turn on either single spin state. And as a result, you into to in the case of uterium, you have no incoherent scattering and the case of hydrogen, you have a lot of incoherent scattering from the two spin states of hydrogen, and that contributes a background, a gray background, and it restricts the accuracy with which you can measure these spots, both where they are and their intensities. So just to illustrate what happens when you do to rate or crystallography, you turn it all to yellow. I think it was yellow wasn't it. Yeah, yeah right so it goes yellow. You put it into D2O solvent and you get a huge improvement in the visibility of the spots. And that means you're going to measure them all better. It means the quality goes up, and it means in fact that you can use smaller samples. You gain in signal to noise at least you can compromise a bit. So that typically ends up with a gain of about 10 is always estimated about 10 in the crystal volumes that are needed. There was also, I think Frank mentioned this as well. He would have measured he probably would have mentioned that hydrogen has a negative scattering like now the reasons for that we don't really need to go into you probably would told anyway. So when you have a hydrogen atom, it appears in density maps as a negative peak. And there are situations where the negative peak can start to cancel out some of the surrounding positive peaks from the rest of the structure so you have the so called density cancellation effects that tend to interfere with the interpretation of the of the maps. And whereas if it's all deuterium then you get this huge visibility which is I showed you before in these maps you see these strong peaks. And so on and there are other advantages as well that you get you avoid nasty absorption effects. And so on. So so that's that's the duration because you want nothing you want no hydrogen left before I do the you just want 100% you don't want any action anywhere. That's the ideal world. Of course you have to be careful because we have to be wary of, of, of isotope effects, and we have to be careful to know that the structures that we're looking at in deuterium form are actually reflecting the real molecule and typically we did that with x-ray you take the D form you take the H form you do the crystallographic study side by side and you say what's different. And the same would be true for solution work as well. And there are isotope effects you just have to be aware of it and you have to do all the relevant tests to make sure that you're not a victim of it and benefit mostly. Now I doubt the one I watched the example I wanted to show you in relation to this is about actually about amylated genesis in a particular protein called trans tyritin. And I love this example because it's got it's got all sorts of different combinations of different techniques as well as having had being lucky enough to have worked with some amazing people in doing it. So trans tyritin is the protein that transports thyroxine in, in, in important hormone around in the bloodstream and in the cerebrospinal fluid. And I, there's a little movie I've been using for a little while now for those of you who don't really know about trans tyritin or amyloid or anything. This is the human being, obviously, and this is an attempt to summarize. It doesn't look like this other movies working. That's brilliant. Right, the movie's not working. All right, anyway, so I'll attempt to, to, to. That's amazing. Maybe there's an arrow here. Okay, I apologize, my movie has not worked. So anyway, so that the trans, the trans tyritin is produced mostly in the liver. And, and it's a tetramer. It's a, I don't know what my next slide is and say, oh, goodness. So it's a tetramer so it's a four, it's a four, it's got four bits to it and it has a cavity in the middle, which is where the natural ligand with thyroxine binds. And, and, and it, and it circulates around and delivering and transporting thyroxine. So it's obviously very important for the endocrine system. And, and, but the problem with it is, is that the tetramer is intrinsically unstable. And, and, and it tends to fall apart and when it falls apart, it sort of falls apart and it reassembles as a federal system. And, and bombs these, these, these amyloid plaques, which in the case we've been looking at occur mostly in the heart, but they can affect the brain they can affect the cause neuropathy it can get into the intestine to all sorts of places. There's all sorts of different mutants that go into different places, even though the mutants are very subtle single point mutations can end up with these things accumulating in, in very specific ways in different tissues. So that alone is quite amazing. And just to give you a feeling for that apologize about the movie again this is a collaborator that you see L have picture from him. He, he, he had a patient this guy Mark puppies who he had a patient who one of his patients with had cardiac, cardiac myopathy from amyloid, and in the end I think she had she had a heart transplant. And this is he took a picture of the heart as it, you know, after it came out. And you can sort of see from this picture and from the other pictures here, they're restrictive nature of the amyloid deposits. If you look at that heart, you can, it literally looks as though it would bounce. It's hard. It's not flexible. It's very restricted in what its function is capable of doing. So that's just to give you a feeling for the real clinical consequences of amyloid accumulation in cardiomyopathy. And of course it's the same in other tissues as well. It's just that you really, it really is evocative. It really does sort of stand out in the way that you understand it when you just imagine how much it restricts movement and pumping. Anyway, so here's the molecule. It's a tetramer as I said for orbits to it cavity in the middle which is where the ligand would go. So the monomer, you can see the monomer you can see it as diamond and of course the diamond which makes the tetramer. And what we did, and this is the same sort of summary. So there's the binding pockets, the different parts of the tetramer and so on. And what we did was I get this won't work either, I guess. No, it's not working. That's supposed to be moving as well. This is a list of single, mostly single point mutations of trans-tibetan, which result in all sorts of different mutations. So this is the reported phenotype in this column here. So polyneuropathy I guess is PN, H means heart. So specific single mutations end up with the trans-tibetan consequences of pathology being very discreet or amazingly discreet. So we were mostly interested in, that's the key. So it affects eye, heart, kidney, different mutations, wild type, and some of the mutations are actually positive, right? They're protective. So there's one mutation that is actually highly protective and it prevents the thing, mis-folding, bombing, amyloid. So that's a very important reference in looking at the results from the more interesting pathological ones. And so what we decided to do was to look, firstly, at highly protective, and that's the T119M, I don't know where the M's gone, but anyway. So, and then there was an S52P, that was the strongly pathological one. So highly protective, strongly pathological, and then the strongly pathological one bound with a drug that is the only drug actually around, that is prescribed for it, that is approved for this. And so you can see the drug sitting in the middle of the structure there. So what we wanted to do was compare all these things and to see if there were any clues in making that comparison. And we realized that there's a lot of X-ray information around at the time, but it hadn't given the answers. So we thought we would do it with neutrons because we thought there's a strong chance that there will be a lot of stuff happening. Under the surface of the X-ray work that could be interesting and it turned out to be exactly the same. So there's the stable mutant, the point mutation, this is the one for the unstable mutant. And so just to give you a feeling for in vitro, what happened. So, and I would just concentrate that we've got all sorts of things, denaturing gels, non-denaturing gels, and here. But if you just compare, look at the right hand row column and this one in the middle and don't worry too much about the other side. So it's basically one of the very unstable mutants, which we call a duplication mutant, but one of them. And this one, this column relates to the stable mutant. And, and what's happened here is that day zero, they've just been left. We've got various things. We've got some stuff left at four degrees, which is some stuff left at 37 degrees. And we've got electron micrographs at various stages along that pathway. So if you look at protective mutant, for example, along this column, you see absolutely nothing changes throughout the whole period of that experiment. Whereas if you look at the pathological mutant here, which the D means it's deuterated, but you'll come back to that. So, and you just see these things just accumulating these, these deposits. And then at the very bottom we've got what happens at four degrees, which is largely because there is some evidence of, of, of thermal formation, but obviously much reduced, which is what you'd expect. And here the H and the D are just comparing the hydrogenated and the deuterated analogs of the same protein. I'm not going to go into the into the gels, but they're also very interesting. Because I think I'm running a bit on time. Anyway, so, so, so that just shows you a little bit about what in vitro what you see and what it is that's slowing up that heart and stopping it in moving. And then when you actually do the neutron crystallography you what you find is, it's nothing short of amazing really, because what happens is you, you have this mutation. You have that this is the protective mutant here and you see you've got a hydrogen bond here, which, which is, which actually has a huge effect on the stability of this loop of one particular part of the protein. And in the, in the mutation, it's, you've got that hudgeon bond is lost and you end up with a loosening of this turn in the loop, which then loosens the entire loop and renders it much more unstable one hudgeon bond. That's what's going on it seems. And when you guys, okay, this is another movie. This is really annoying. Anyway, that should have started anyway so so. So anyway, what that was designed to show you it showed the molecular dynamics calculations for the monomer alone the diamond alone and the tetra alone and what you would see if it worked, it would be that the monomer alone would fall apart much more quickly than the dimer which in the monomer fall apart more quickly than the tetramus of the tetramer forms a very important part in stabilizing in stabilizing and falling with the monomer falling apart so it's a, it's, it's the constraining factor if you like. So, and then there's some principle component analysis that shows them, this is the loop in question. But it's not read where it's most mobile. And that is basically graphically showing the bit of the protein that is most unstable and the heart problem. So, and then this is where the drug goes. And what you see is the drug pops into this area of the structure it recruits a water molecule. And in the end, the most amazing thing is if this is the unstable with the drug. It recruits this water molecule into this place and in doing so makes it look almost exactly like the stable mutant. So it's doing something closely analogous to that it's not the end of the story because the drug is not actually universally successful. It doesn't sometimes that doesn't work but there's a very interesting insight there and became an important part of the story. So we get back to I'm going to finish off really about making gaps gaps as well and for this particular project, we are linking up with colleagues at London, the Royal Free. I'm going to finish in one of them already. What we are doing is trying to look at different mutants to identify with the stuff that we produce in the lab in vitro and the same mutants from patients and proteins that are purified because we want to try to link things up and try to make them clear that the experimental stuff at the bench is is relevant and linked to real clinical issues so that that's going on. And we're also looking at different drugs because the only one that exists is not universally applicable, and we're looking at different candidate drugs this is some of them the x-ray structures are some of them were preparing neutron experiments as well. So for example, the bottom that's one of the drugs in binding both parts of the structure so it's going right across the entire church where's the other drug so far is actually linked in one site one side of the molecule not both. And, yeah, so so that's where we are and I'm going to finish with this slide it's old but that's I'm blaming the lockdown for not having a recent picture of this is 2018 or 19. So because they've done a huge amount of the work in developing the facilities in the group that we've got, which is actually a rather lovely group and as enabled and then doda here he came up to the picture he's the electron microscopy guy who runs he runs the cryos with some other colleagues. And I think that's all I've got some of the funders. So I'll stop there and if there's time, anybody can ask me questions. I'm happy to try and answer. So do we have any questions for Trevor here. Let's go back to the contrast to figure. And there are different proportion of different protein. Sorry, you're talking about one of the early slides. Yes. Okay, let's just try. There are many ones and then there is a cross of a different proportion of the one that's one. Yeah, back back to the one, the classical one. So if you go back. Oh, sorry. Right. She won't ask. Yeah, I want to ask if you have different deuterated protein, but what about the solution, the solvent. I mean, how can you control like in the like 0% on deuterated protein that you need to put it into H2O 100% deuterated protein. You need to put it into digital then how can you do like 50% deuterated protein. Because there are some hydrogens will will like interact with the solvent. Yes, absolutely. But you what you have to do is the late relates the exchange to the exchange to the situation that you're aiming for so in the end. So basically we informing recipes for making match up, right, match up proteins, you just have to aim for zero contrast so you adjust your just your conditions, and we have a protocol for doing this, so that the thing matches out at 100%. So even though there is a label part of, as you say the solvent, there is a label part of it. So by label different proportion of protein, we can see different protein in the same. Yes, I guess so I hope I'm understanding you correctly. Yes, you can live I mean this is all statistically random, right. The degree of, and it's the only reason it works is because you're at your, it's solution scattering you're at low resolution you're at sort of 20 angstroms resolution or something. And then you will be suffering the, the problems that, you know, where you couldn't really assume an average scattering makes sense if you were much higher resolution. Thank you. There is from Susana. You have a question. Yeah but actually Jennifer was before me. Let's go ahead. Jennifer can wait. She has all so much now. Yeah, so go ahead. Yes, so thank you for this presentation so I have actually two questions. So first one is about the segmental deterioration or just labeling. Are you like do you have a service for this. Yeah, actually a segmental is something I don't know if I do if I refer to it in his talk because he's actually done it. We haven't done it enormously we've looked at it a lot in terms of we thought originally started talking to Michael Sattler about it because he did a little bit. I think it was in team technology that he used for that. You've since sort of been thinking more about sort is we haven't developed that as a routine as a routine service, but it is, it is definitely something that we'd like to do more of Frank did it with Michael Sattler and benefit. I think he might have even referred to because he had one paper where he used it. Is that what you meant sorry I'm not sure. Yeah, exactly. Because I know. Yeah, they used to sort is a. Yes, something called utilize one. Right. Okay, that's something I would defer to Michael heartline. But I just remember that we started off thinking with it about in teams and then we end up feeling that the sort is approach is better, but it's actually a little bit. But it certainly something we could talk about. Because it is a development we'd like to undertake. Yeah, I mean I will be interested in this as well so also for NMR. Yes, yes. Actually, I mean a lot of these tricks derived from NMR. Yeah, I'm not sure I get the reason Michael Sattler was doing it. He's an NMR person. So that's probably exactly why it came from. So yeah, absolutely. And then my second question so how far did you go with deterioration using insect cells. No, I mean both in both insects and mammalian cells are we meant to get going with in so we haven't done them. But we've jumped mammalian because really it's more relevant to most of the biological problems the insect cells of course are quite fragile. And so they're difficult but then mammalian cells are as well and we basically just jumped straight to mammalian cells and we've only just started in that. And it's not going to be trivial this, it's very unlikely, we're going to there are some amazing developments around that. But it's very unlikely we're going to be able to get to 100% utilization of mammalian cells so I think what we will end up doing, you don't need to get to 100. But the solution is definitely you don't need to get to 100 you just need to get to some level where you have good contrast. And then if you get a contrast difference that amounts to 30% solvent, then that's exploitable. And, but it's very unlikely that we're going to get to something with extremely high levels of or deterioration with mammalian cells. So we realize that but you then to come up with different match out regimes and how you're going to use it and work out all contrast and so on, and that will be done, you know, experimentally testing. Yeah, well because I actually have a system which I can use in insect cells because there's no glycosylation or so, but I can't do this in E. coli so that's why I was interested in insect cells. Yes, right. Okay, yeah, right so it's the same reason that you were, but when you was it a was it what sort of protein was it was it was it. Yeah, so it's a do you pick with names. I see. So it's relevant to to mammalian systems. Yes. Okay. So, so then you have to the risk is that the glycosylation might be different, I suppose. There's no glycosylation actually. Oh, there's no glycosylation. Okay, okay. So what was your points that that you did it in, you didn't do it in Coli. Now it's a bit too large in E. coli so it's going to. And then maybe the last question does have a curiosity about the TTR. So the structure which you showed in the, or the small molecule. Did you test also other small molecules and did you see also this. Sorry, I'm just going to. You mean at the very end. Yes. So this connection with water. So if you said also in different small molecules with the TTR. You mean this bit. Now I think the previous one actually this one. Yes. So the TTR has two, two, two. That's the drug that is currently approved. And it, the tetra has two binding sites and this is a monovalent drug. And because there's some sort of negative co-operativity effect going on. When you put get one drug in, it tends to affect the other binding sites that nothing binds to it. So the hope is that we are hopefully hitting both binding sites. They are with one drug so it goes across the gap and it bridges both binding sites. So the hope is that it would be better because of that reason. And so is that sorry, is that what you're asking. Yeah, I was just a bit confused about this. Yeah. Is that makes sense then. Yeah, I will read the paper as well. Thank you. Hi, I had a question about the stealth nano discs. Oh yeah. So I kind of understood from the example that they were used to crystallize membrane membrane proteins I'm assuming for a neutral crystallography. No, no, no, no, it was never, never designed to go to crystallography for us. So it's like sans kind of. Exactly. So it's all the resolution, solution scattering it. I mean, actually what's what's interesting is interesting that you say that because if you look at the, I'm going to go. I really ought to have a list. Yeah, so if you look at those things, yeah, yeah, if you look at these things there, just as they are one of the nice. There's different ways of doing this there's another thing called smelts like this steering my leg acid things that's but conceptually difficult, you know, this crystallization is probably quite difficult but what does happen is that the discs, but sometimes sit on top of each other and form extended structures filaments. And of course, if you can line those up, you can get fiber diffraction data of, I mean I had to give a talk tomorrow about fiber diffraction. So you can actually end up with filaments that you could conceive of the study by fiber diffraction but they're certainly further for the neutrons I don't see crystal like crystallizing that being feasible. Who knows, never say never but. That answers the rest of the question.