 Thank you, Tommy, for the introduction. And then, Tommy, for the organization, it's the program is really amazing. And I hope that the students find different, interesting topics for their future. So as you said, in Grenovo, and in the FEDID image, you can see a neutron reactor on the synchrotron. So we are in a very appropriate place for structural work. Just to introduce your problem with this place where I work, I am, in fact, on the compass of the city. So it's the other side of the city compared to the neutron in the institute that was on plant polysaccharide. But now it's more generally on glycoscience. And we have some collaboration with our colleague at ILL on the SRF, and I will present some work that we did with the colleagues at ILL today. So first, about carbohydrates, I just pop up this morning to hear a little bit about Marta. And she was talking more about polysaccharide. So this is one side of carbohydrate. You have all the energy on structure, pectin, cellulose, starch. And then you have the other side of glycan, which is more what happened on cell surface. So as Tommy said, there is a lot of study on what happened into the cell, cell assembly. But it's very important also to consider what happened at the cell surface. So all the work on lipid, membrane, but also what is to the external part, which is more a sugar. So when you open a book sometime about cell biology, this is what you see. And you have a perfectly smooth and clean cell surface, which is not at all the reality. Because in reality, you have something sick on all cells. It's universal. All cells are covered with this sick, sweet stuff, which is, in fact, the glycocalyx. So it's an assembly of different sugar, exopolysaccharide, glycolipid, glycoprotein. And everything that arrives to the surface of the cell will be exposed first to sugar. So you can imagine that this will be the same thing as comparing a dessert. So we are not a dessert. Our cells are covered with this kind of bushy stuff. And we call it the glycolandscape. You can imagine it as a forest. So what are these glycolandscapes made of? There are glycolipids and they present branch glycans. So glycans are different from protein and RNA. They are branch structure, complex structure. There are also glycoprotein. So glycolipids are represented here. Glycoprotein also have complex glycan at the surface. On there, they can be also extracellular stuff like decosaminoglycan, epahan, and other complex polysaccharide around. But everything is covered with this. To have a more molecular view, this is what we are looking at. So glycoprotein that have, in general, N glycan. So link to asparagine, why are they here? Asparagine, who is a very well-defined branch structure and then complex sugar at the surface. Or glycans that are linked to serine or trionine. So this is well-defined on protein. They are more rare glycosylation, but I will not talk about it. Glycolipid, they also have some linear or complex sugar. And you can have some more complex glycoancor, glycoprotein, or more complex that are appearing now. What I want to show here is that some sugar are always at the surface, some monosaccharide. And this will be the one that are mostly interacting with, let's say, for example, pathogen or other cells. And here is a representation of monosaccharide with symbol, which are accepted now by all the community. And today, I will tell you about recognition of cyallic acid, which are this violet diamond. So accessible at the surface, very often. And also fucose, which is a small sugar here represented by your red triangle. So they are important in this case. We know that glycosylation is important because if you perturb it, for example, you knock out animals, knocked out mice or other, quite often you don't have animal at all. So they die in the embryo state. You need glycosylation to live. So if the disorder is tiny, for example, only one chromosome affected and not both of them, then you will have congenital disorder of glycosylation. And now there are quite a range of disease in humans that are known to be congenital disorder of glycosylation. They are very severe. Very often it's kids who are very deficient mentally, physically who cannot live very well. So there are not many cases because this is nothing that will spread in the population as a genetic disease, but it's help also to understand the importance of glycosylation. So this is one sign that it's important. Also now it's accepted that all the chronic disease, the ones that are really burden on our society, cancer, diabetes, let's say neurodegeneration disease, inflammation, they are related also to carbohydrate. You can associate them with a modification of the glycan on the surface. So sometimes it's a correlation, sometimes it's a cause, but in all cases there are problems. So all these big diseases you can associate with a problem. But today I will talk only about relation to infection because since we have all these sugar at the surface of the cell, they are very good target for microbes. So this will be virus, fungi, bacteria, and they can really use our sugar for addition on infection. I give one example here about some glycan that do not seem to be related to else and they are the blood group. So the blood group they were discovered, you see 150 years ago by a lunch tiner even as a stamp on a Nobel Prize. And he discovered that, he discovered the blood group system and it's only 50 years later, no 100 years later that say it was realized in fact it was not a protein epitope but it was a sugar epitope. So your blood group consists of a small oligosaccharide as a surface of your red cell. In fact it's also present on other cells, endothelial cells, in your mucous, in your saliva for most of the population. So if you have this yellow rectangle here, this is a galenac, your blood group A. If you have a yellow ring, this is a galactose on your blood group B. So just a change of an acetyl group on galactose, your blood group B and A. And if you have no galactose or galenac, your blood group O. This is very simple. And it seems that the only problem that it can create is a problem of transfusion because if you have blood group B, you cannot have someone giving you a blood group A, et cetera. So at the first glance, it's controlled by one allele, one enzyme, which is present here, the different enzyme here and no enzyme here. And let's say no effect on the population if you don't need a transfusion. In fact, in this case, it should be over expressed on all the population at the same way. But in fact, it was studied that other variation and the variation have been very well studied by Japanese people. Very interested in blood group. And it was discovered that, for example, in Europe were between A and O, a little bit more A. UK is a little bit different, a little bit more O. Africa is mostly O, whereas this is particularly true also in South America. So Africa is, let's say it's majority of population in O, but South America it was completely O before, before let's say some people arrived by boat from Spain or other. So the European people, they traveled across the North of Russia and they arrived in Canada and then these lands were populated by the North. And it seems that something happened at the Mexican border or before and there was a change in the population of blood group. So the sugar depend on something. And what we realized later, some people realize is that it's related to infection. In fact, in Europe, for example, there is almost no blood group B and it seems to be related to the black plague in middle age. But this is at the moment well studied, but not proven. What is completely proven is that cholera, let's say everyone gets infected by cholera, but the disease that can really kill you, the form that can kill you, it's more sensitive to people with blood group O. So blood group O has been removed slowly. This is epidemiology study in Bangladesh and in this part of the world. And in Africa, it's malaria that gives also the most bad cases. So let's say neurological cases of malaria with people with blood group A. So we realized that in fact, there is an influence of blood group A on the health of people, but this is related to infection. So what you can understand of importance at the moment is that the sugar that we have on our cell as many other things, in fact, there are the result of evolution. They have been a co-evolution between animals or between all organisms on the pathogens and there is a pressure of the pathogens to clean at some moment the evolution. And in fact, it has been even proven recently by French people, they were studying rabbit. And the rabbit can have hemorrhagic fever, which is pretty bad. In fact, this study was funded by the UNTER, UNTER Association. They wanted to have some rabbit and they studied that. And they realized that the whole population of rabbit with blood group O, I think it was, was just removed in few years by a bacteria. Was it a bacteria? I think it was by a virus. And few years after, slowly they reappeared. So there is a direct influence of infection on our blood group. And the role of glycan on our cell in this case is to create diversity. So when you have a new bug appearing that will recognize one sugar, it will not kill the whole population. Some will escape because they have different sugar. So this was about the importance of variety of glycan. It's complicated on a biochemical point of view, but it's very important for us. So if there are glycans and if the pathogens or other cell recognize them, there should be a way to read the code. So we call this complexity of glycan the glyco code. And we need something to decipher the code. And this code is recognized by enzymes. So you have the enzymes that make the sugar. You have the enzymes that destroy the sugar, glycosyl hydrolysis. And you have some proteins that are not enzyme. They are not antibodies, but they are receptor for the sugar. So they can decipher the sugar constitution and they can pass the message to some something else in the cell. So you can find them on attached to membrane on different cells. So these are lectins that are embedded in membrane. And you can also find some more circulating in our body. Some are associated with toxin and they allow toxin to enter into the cells. So they can be associated to internalization. And some are located on the surface of pathogens. And if you're interested, we have a database unilectin surgery with all the structure of all lectins, which are other beautiful. So in the case of pathogens, there are many pathogens that have lectin on the surface like staphylococcus, cholera toxin also is associated with lectin. And I will talk a little bit about pseudomonas and influenza. And we are interested into that because in fact it's a way to fight against infection. If you can lure the pathogen, if you can give it a mimic of the sugar instead of giving it the real cell to attach, then you can prevent the adhesion. It's not an antibiotic, it will not kill the bacteria or the virus. So there is no pressure on the resistance, but it can help with antibiotic treatment. And there are already some cases, for example, for urinary infection where you can do this kind of treatment. And it's a natural way to protect the babies with human milk. Human milk oligosaccharide, they have several effects on the baby, but one of them is to prevent infection by let's say competing with the sugar in the intestinal tract. So I will give you an example with influenza. This is not my work at all, it's rather old, but we always look for example where structural biology can bring solution for drug and also glycobiology. So influenza is perfect. Influenza, this year it's very quiet, no flu case because we have COVID and we have mask and we hope that next year the flu will not be back. It seems that several strains disappear, so it may be good news, but maybe it will be a strong flu season next year, we don't know. But I mean, it's a very deadly disease, especially when you have this H1N1 or other dangerous strain. The virus has two proteins at the surface, two main protein, one is emagglutinin, HA, and the other one is noraminidase Na. And emagglutinin is the lectin, so there is a lectin at the surface, it attached to the sialic acid at the surface of our cell, it attached and then there is the entering of the virus in the cell. Noraminidase, it's the other role when the virus after multiplication get out of the cell, the noraminidase clefs sialic acid, so the virus can swim away from the cell and disseminate. So you'll notice that we have H1N, and you know that the strain of flu or name H something and something, and in fact it's because of these two surface proteins. So influenza, I will talk about influenza A, influenza B and C, these are human mild infection and what we call the season flu, it's not of importance, let's say, therapeutical importance. All the other one, they have a type of emagglutinin and the type of noraminidase, and they are associated with either a human, pork, poultry, or even bats or seals, but you see that in a case, the problem is that the case of avian pig and human, there can be some species that can move from one species to some strains that can move from one species to the other, and this is a big problem because they can mutinous species, come back to human, et cetera, and you have the very dangerous one, H1N1 Spanish flu, H5N1 avian flu that can be dangerous for human, and this is based on the sequence of emagglutinin on noraminidase. If we go to the structure of emagglutinin, so as I told you, it's there to enter and fagocit in the cell, and it's a timer, it has been solved quite a while ago, work at EMBL, we stick to that also, and it has three sites for cialic acid at the top, the ones that recognize human cell. The importance of emagglutinin, it's because it's involved in the species barrier. This is the ones that decide if it goes to a bird or to a human. So on human receptor, you have these galactose on gluconac, and you have a cialic acid that comes on the oxygen ear position c-sogalactose. In bird, you have another gluconac, cialic acid, so it's not at the same time, and it goes on position three. And just this difference make some strain attaching to human, of my French slide, on some strain attaching to bird. So just by looking at the difference, we can say which one is which. And there have been crystal structure, and this is quite of importance because in fact, there are two amino acid difference, so human H1 on H3, on these two amino difference, they can switch from a bird emagglutinin that attached to cialic acid position three, on the human emagglutinin that attached to cialic acid position six. So this is quite of interest for the people following the mutation because they can see at which moment a bird flu can become a human flu and be very dangerous. Noraminidase, it's another story, but against our sugar, in this case, it's an enzyme. It's tetrameric. And if you look it from the top, you have this foresight again for cialic acid. And in this case, quite buried, as you see, in this case, that's Australian crystallographer who solved the structure in 92 when they could decipher the binding to cialic acid and also they had a view of the transition state of the enzyme. Just from this crystal structure and all the know-how they had resulted the only drug that we have against flu. So you see that the one that is called in France, tamiflu, the chemical name is osteoamavirinivir and the other drug, anamivir, and that may be all Europe, I don't know, they have been designed from the structure of cialic acid in the binding site, maintaining all the hydrogen bonds and hydrophobic contact of importance. So this is really the success story for glycobiology, structural glycobiology, that if you have the crystal structure with a substrate, you have a chance to design a chemical drug that would be of importance in case of serious infection. So this was to convince you that protein, lectins are important, also of importance on our glycan and that we should study them at the molecular level. So now more generally, what are these contacts that we want to study at the molecular level? How protein do recognize sugar? So lectin, as I say, they are not enzymes, they are non-covalent, specific and reversible. They can bind to monosaccharide and oligosaccharide and the affinity is not antibody affinity, it's not nanomolar. Generally they say, well, lectin bind to sugar only to millimolar range, it's not true for lectin from pathogens because of this co-evolution, they are quite efficient and they bind to micromolar range. It's not huge, but in fact, as you saw with this trivalent emagglutinin, they are multivalent. So because they are multivalent, they have a very strong avidity effect and they stick to our cell in the quite strong effect and it goes to nanomolar because of avidity, but each site is more in the micromolar. The forces involved are at first to say hydrogen bonds because sugar is just hydroxyl group that are presented in a different way. If you have a galactose, a glucose, a manose, it's a different scaffold, but the scaffold is to present hydroxyl group OH. So hydrogen bonds are very important for the contacts between protein and sugar, protein, yes, protein and sugar. There are also hydrophobic that has been recognized as very important also now, like for example tryptophan or tyrosine, so aromatic amino acid that stuck to the CH phase of sugar. And this also creates affinity and it also creates specificity because if you have a sugar with OH here instead of hydrogen, it cannot bind. So it makes specificity, quite important. That's what we call also CHP stacking in this case. In many cases it's not represented here, but there can be some bridging water. So if you can have a hydrogen bond that goes through water and then to an amino acid. And in several lectins, now we also observe direct involvement of metal ion in 95%, almost 99%, it's calcium. So calcium can be coordinated by the protein and by hydroxyl of the sugar. So because of this, because of the kind of contacts you see immediately that we are interested in hydrogen. Hydrogen atoms, they are the ones that you cannot see in X-ray unless you go to 0.9-angstrom resolution, but even in this case. And they are the ones that make the contacts. That's also the ones that make the hydrogen bond. You need an hydrogen, the ones that make the CH contact, the stacking contact with aromatic. So we are really interested on where are the hydrogen bond, how are they involved in the binding. And here, this is the moment where I switched to neutron because neutron is what you need when you want to see hydrogen. Maybe you will not see the proton, you will see the deuterium, but they are all hydrogen atoms, the family of hydrogen atoms. So the advantage of neutron crystallography compared to X-ray crystallography, and I love X-ray crystallography, but the advantage is really that you will see your hydrogen. You don't cook your crystal, so you can use it for something else compared to X-ray. And you will see that it's important also that we work at ambient temperature. So X-ray, especially at synchrotrons, they are so powerful. You make your measurement in two or three minutes instead of one week at neutron, but then your crystal is completely cooked first, and you have to freeze it first because you will not survive even the two minutes if it's not frozen. So advantage of neutron. Limitation, well, it's slow. So as I told you, you need one or two weeks to get complete diffraction data on one crystal. You need big crystal, but this is not so true anymore. And I will show you example with reasonable sized crystal, and you need time. Because of that, there are not that many neutron crystal structures. This has been reviewed recently by our PhD student. We would defend the thesis. So there is only 180 neutron crystal structure of protein. And among them, there is only two lectins co-crystallized with the sugar in it. So con canavalin A, a plant lectin, and gallatin, a human lectin. But there is no, there was no bacterial lectins. So we want neutron, but we want also something more. We want to do the neutron structure with the deteriorated sugar in it. We know that you can deteriorate protein. I will not come back on it. I think you had some talk about this. What has never been done before is to deteriorate also the sugar ligand. And this is an example of, in the two example I told you, galactin-3, it's a beautiful crystal structure, which has been made in Lund. They use a human galactin, a per-deterrated, they produce a per-deterrated human lectin. But they use lactose from, just from milk, not normal lactose that has been exchanged with deuterium. So this means that you can see the D on the OH, but not the one on the carbon. So I'm not a neutron physicist. I will not come back to why it's important to exchange hydrogen on deuterium. It has to be related to neutron coherence scattering. And I think you heard quite a lot about it. But exchanging the H on the OH is not quite sufficient if you want to have a complete view of your sugar ligand. We also want to have the deuterium present on CH, like the CH of the ring and the CH of the metal group. It's not only because you don't see them if they are not deteriorated, but the signal is also canceled because of this neutron coherence scattering and incoherent neutron scattering. I cannot explain you why exactly, but having hydrogen, in fact, it destroys the density around. So it's really a problem and we want to have it to solve the problem. So now for the second part of the talk, I will present one example of this kind of work that we did with Lukash Gajdos, the ILL-PHD program we defended a few weeks ago. And we did excellent work on two bacterial lectins. I will just choose the one on Pseudomonas. And this part is not published. I'm sorry, but it will not be on the site unless the paper is accepted soon. So Pseudomonas Eruginoza, we are talking now not about a virus, but a bacteria, but quite a nasty one. It's what we call infection that are not bad for LC people. You do not have any risk if you're in good health, but if you're at hospital, if you're immunocompromised or if you are cystic fibrosis patient, then you can really have a severe infection of Pseudomonas. It's everywhere. It's in hot water. In this case, the dangerous liquid is not the champagne, it's hot water. You can really leave some dermatitis and problem with Pseudomonas Eruginoza. And now it's on the antimicrobial resistance list from the Wu Organization, the escape list. It's in the most, six most dangerous pathogens in term of future. Because in the future, there will be more and more antimicrobial resistance. And when you get an infection, Pseudomonas infection at the end of the hospital, you can really have a problem. So it will be important to have a way to block it. Pseudomonas Eruginoza has a real strategy to bind to our sugar. It has pilli that bind to some sugar, but we have problem at the moment to study which one. It has one flagellum with the extremities that seem also to bansaialic acid. But what we know is that it has also soluble lectins that are made into the bacterium, goes out and helps attaching to substrate. And leg A, oh, it's inverse here. Sorry, leg A bind to galactose and leg B to fucose. I have to change it. But I will talk about leg B. So leg B is a fucose-specific lectin so it's started made by Pseudomonas, present then on the surface and ready to attach anything with a fucose on it. It has the particularity to have a binding site with two calcium bridging the fucose. So these two calcium, they sit on the protein coordinated by many acidic group and they coordinate three hydroxyl of the fucose. It's very unusual. No other lectin does that, no other bacteria do that. So we have some things that is quite unusual and can serve for designing drug. So this is what is known from crystal structure. As you see, no hydrogen nowhere, only carbon, oxygen and nitrogen and we want to know where are the hydrogen. The question that we have is what is the directionality of hydrogen bond? Which one is from the water? For example, a roll of water molecule and which one is attached to the OH? What is the protonation state of the acid because there are six acidic side group in the protein and we want to know which one is protonated and which one is just CO minus. So there are three questions that can be answered by neutron and cannot be answered by X-ray. The methodology is classical, produce deuterated, adapt bacteria to deuterated medium, produce protein, produce sugar, per deuterated, first per deuterated sugar, purified crystallized neutron. Very classical, except that it can take 18 months to do this and another 18 months to obtain the crystal. So they are pitfall at every step when you start this kind of work and you don't know how much time and energy each step can take. So structure solving is finally the easiest part. So I will tell you a little bit about per deuterated sugar because this is quite unusual. As I told you, there was no crystal structure of per deuterated sugar and in general there is no crystal structure of per deuterated ligand in protein. So you can say, okay, I would just buy it but in fact only glucose and mannose, very usual sugar, the very common sugar can be bought as per deuterated and it cost about $1,000 a gram. And once they do it, in fact, they grow it from plant. They have plant growing them in D2O and purify glucose or mannose. You cannot do that for glucose, our sugar. You can do chemistry. This is hardcore chemistry. You have to exchange all hydrogen one by one. You need a real good chemist and you will not get one gram at the end for sure. So we turn to synthetic glycobiology which is to convince bacteria that they should produce deuterated sugar. Problem is that sugar, they are not the product of one gene like protein. You have to get many genes to assemble them to have a wool plant in a factory in a cell to get the sugar you want. So in this case, we turn to someone in our institute who is working on producing human milk oligosaccharide. So this guy is producing this fucal fat two gal beta one for glucose. So it's fucosil lactose. So lactose with a fucose which is very common in human milk for women and protects the baby from infection and it's not present at all in cow milk and it can produce gram by engineering bacteria. And this is something that is now commercial and you see this, I think you cannot buy it in France yet but in US it's commercial now. So it's milk with human milk oligosaccharide that have been made in bacteria. But for this he had to engineer a pathway so all the green enzyme have been inserted in the bacteria and he gets this one. And what we want, we want fucos. So in our case, he did another modification he inserted a fucosidase. So this is what has been inserted and he dilated some enzyme for example the ones that transform fucos in fucitol because fuculose because we don't want fuculose or the ones that will reintroduce fucose because we want the fucose out in the medium of culture. So from this, the students did the production so months to get the bacteria accommodated to D2O because all bacteria don't grow in D2O you have to get them accommodated and then it got the production of per-deterreated fucos and you see that you get about 10 times less than normal fucos because bacteria are not in really a good shape but they produce and then you have to do the mass spectrometry check that you get what you want you have to purify fucos from lactose and from IPTG that is put in the, as let's say to push the culture and do NMR and you can do NMR of proton or you can do NMR of deuterium and you check that you have your compound. So 18 months in total because this is something really inovant to produce this. Up, what is doing there? So okay, I did not present the proper button. Excuse me, revise everything now come back to fucos production. Fucos, here it is. And now just with images you need all the batch production. You need very good team in term of purification because this is quite analytical work. And at the end, he was happy that he got 220 milligram of luffelized de-fucos. So enough to run a structural work. From this, it seems almost easy to have the production of the protein, so deuterated protein. In this case, you see that you can get 200 milligram of deuterated, per deuterated protein. Gross crystal, again, this can take weeks and months. And since we want to do neutron, we want big crystal. They are not that big. You see they are about one millimeter long on a zero, three millimeter large. But that was big enough for Danny. So we started with this crystal. So once you have the crystal, you can, you did your application. We can work to discuss about application to neutron. But once I see the beautiful crystals, I agree. So in this case, we work at Lady 3 at ILL, the Laue apparatus. And then you have a series of different experiment to run. First you get your neutron data collection and you can do the reduction of the data. On the same crystal, you go to a synchrotron or better to a classical diffractometer because then you can also collect at room temperature. So it's better if you can have neutron collection, X-ray collection at the same temperature on the same crystal. So better to do the neutron first and the X-ray then. And then you have to use special software like Phoenix to do a core refinement with all the data from X-ray and neutron. So this is the Lady 3 beam line with Matthew Blackley who has at ILL and I'm not a specialist just to tell you that it's quasi Laue and there are now a new detector that allows to work with smaller crystal. So finally you don't need several millimeter if you have a little bit less than one millimeter in different, it's okay. So that was our chance in this case. So this big crystal that I show to you, the one that is one millimeter on zero three and you see it's quite dirty around but we could fish it. Finally, it was not good. When we collected the data, it's the most ICT is too high which means that there is some disorder in sight and you see that the spot are not clean. They are elongated spot and since we are doing Laue so multi-wavelength this is something that you cannot choose. So what happened is that Lukash if on another crystal that looks better and you see it's much smaller, zero six by zero four. So something that we could not do a few years ago doing getting a neutron diffraction on this size of crystal. This is almost crystal that you have for doing X-ray. So it took this crystal and in this case we had a beautiful Laue diffraction of course it took us two weeks to collect because small crystal longer time but we could collect the data. And from this I pass all the refinement part. So this is the core refinement of neutron and X-ray. This is what we get for X-ray. So this is the electron density of X-ray is the Ronin angstrom for the X-ray. On X-ray it's high resolution. So you see the sugar very well. You see the hole in the middle of the ring which is beautiful for X-ray crystallography. Calcium are big, they diffract a lot but when you add the neutron density you have more information. Well for the calcium no because calcums I don't, because of the scattering I don't remember exactly they're small in terms of electron, in terms of neutron density but we could see all the deuterium beautifully. So we see now where the OH wants to point so we will see the directionality of hydrogen bond. We see how the metal group is oriented and we see also all the CH group on the ring. So this is a quality that we obtain by using deuterated fucose. So the question that we had was first where are the hydrogen bonds? So you see now we can see the directionality of all hydrogen bonds to acidic group. So we could see the OH exactly in which direction and this could be of importance for designing mimics of fucose for drug. And we could also answer the second question which was is there some protonation of acidic group in the binding side because we have six acidic group so minus, which make a charge of minus six. So we have two calcium, so calcium is plus four but still it's very acidic and the answer was no, none of the acidic group is protonated. So it was also important questions that we could answer by looking precisely at the neutron density. We could also have some idea of the reason for the high affinity of leg B for fucose. Leg B has affinity below micro molar. It's very unusual for a monosaccharide and there is one reason is the presence of calcium and let's say the delocalization of the calcium charge around. And in fact, we could understand that by superimposing electron density and neutron density, we could see a continuity of density that explains that the charge goes to the oxygen, the hydrogen and then to us party group on back. So there is a complete delocalization of charge and we have several of these things that explain the high affinity. The calcium are really circulating, well, they are not circulating electron they are circulating the charge around. I told you it's important to have room temperature and in this case, we have the X-ray neutron refinement that has been done at room temperature and we have a cryo X-ray. And when you look at the B factor, so the structure but with some idea of thermal motion, we first of interest, we can see which group are mobile and which group are not mobile. So it gave us also some view of the dynamic of the protein. I would say that teens are quite rocks in general, they don't move, the teen is six. So there is some movement but not much. But in this case, it was of important because you see here that you have three loops that seems to be quite in scale manner. So if one move, the other one will move and the other one above also. So there could be some concerted movement in some area and there's some things that you can see only with room temperature. The other thing that we could see at room temperature is the dynamic of water molecules because in crystal structure of protein, sugar interaction, it's always said, well, there is a bridging molecule, it's a structure water molecule, it doesn't move, it's here. And then we realize that this is true, for example, for the water molecule here. So these are the four sites, the four units, asymmetric units. So they're all independent. And there is one water molecule, protein-protein interaction here. It's really structural, it never move, change a little bit, but it's always present with the same hydrogen bond. Whereas the water that are involved in protein carbohydrate bridge, they move a lot. They are never at the same position, they rotate and they change hydrogen bond. So the idea of completely fixed water molecule appear to be wrong in this case and it's of importance also for the affinity. Something I forgot to say about the advantage of room temperature is that in another protein, another lectin, when you go to cryo for synchrotron, you use glycerol. And glycerol, it goes into your sugar binding site, it creates problem, it goes in the loop also on the side and we realize that the room temperature that are done without glycerol were much more closer to the physiologic states than the one with glycerol. So room temperature, you avoid glycerol. So this water molecule story, it seems to be tiny, but when we superimpose the neutron structure that was done without, now the X-ray structure was done without focals. So that was done by another team. They see water molecule exactly where we have the oxygen of our focals. So these water molecules, they are pushed out of the site when focals come in, they are released and this is very good for entropy, so high affinity. On the one here, the ones that stay when the focals goes in, this one, it's a mobile water molecule. It's not blocked by the sugar that enter it. So again, it's favorable in entropy. And indeed, our lectin binding to focals, if you cause, it's one of the rare one where the binding is anthropically favorable. Usually it's enthalpy favored on entropy unfavorable, but in this case, it's anthropically favored because water is just released. Water is happy to go out from the site. So again, something that we could see only with neutron is the dynamic of the water molecule. So just a quick word about thermodynamic because I talked about entropy and enthalpy. What we did also is to measure by ITC, the affinity and the thermodynamic profile for the deuterated, I should have put it here somewhere, here in water on the, sorry. Hydrogenated focals and lectin in water on the deuterated focals and lectin in-ditozo and compare the thermodynamic because some people will tell you while you're looking at deuterated systems, they are not the reality, they are exactly similar. So the deuteration does not affect the affinity in our case, it does not affect the enthalpy and the entropy and what we could deduce from the neutron structure is fully relevant to the biological state. So I think I talk a lot and I wanted to show you just because you have some exercise to do about putting an application. So this is the kind of application that we did at the end, we did not fulfill this one. It's continuing with oligosaccharide, but just to show you that if you write your application with stating your case, how important is it? Showing big crystal because the committee, they love to see that you already have crystal of good quality and then explaining clearly why we need neutron for this project, then you have all chance that your application is successful. Just to tell you that this was done so mostly by Lukash and I have to find Lukash is here, Slovak student at ILL, also he was working in the group at ILL with Trevor here and where is Juliet somewhere here in the back and with us at Sermav and he has to go through the city quite often but he was mostly at ILL and here again and all the group involved. And I'm ready to listen and try to answer to all of your questions. Okay, thank you very much, Jan, for a very interesting presentation. And it really points on the uniqueness of the sugar. I mean, it's amazing how nature have designed this system which is not controlled by the genes directly. So that is the intriguing bit of it. So that's more to life than DNA, so to speak. So is there any questions to Ann? Please go ahead. Yes, very interesting. I have some questions. You say that the glycan and the protein have interactions and you say when we have calcium that kind of interaction is more stronger. And what about other ions like an A or something else? Why is calcium important in this kind of interaction? Well, this we really don't know. I think we only observe bridging calcium at the moment. In some case, like conchanavelin, I have also a manganese but the manganese is more to maintain the protein architecture, it's not bridging. You can replace the calcium by something else. You can replace it sometimes. I mean, in the lab, you can try to replace it by manganese. Sometimes people, one of the lectins, we replace it by olmyum to get a signal for synchrotron. You can use it also sometimes. But in nature, and it seems to be calcium. So sometimes you have to be careful also because when people solve a structure, they say why it's calcium and they don't check it. So now there is a tendency in the PDB to say that, well, maybe half of the structures that say we have calcium, maybe it's another ion. So it's always better to check with another method. But in lectin, it has been checked and in general it's calcium. So I have no idea. Maybe the coordination ring is the perfect geometry for hydroxyl groups that are on the ring of sugar. So adjacent hydroxyl group, when two of them are equatorial, and in this case, three of them equatorial axial is just the perfect geometry for the coordination ring of calcium. So it has to do with the property of calcium itself. And such interaction is like to do with the specific amino acid or just general protein. It's mostly acidic amino acid. So you have aspartate and glutamate. You can have a little bit, sometimes we have some other CO, but you always have at least majority of them in the binding site will be acidic amino acid. So yeah, aspartate and glutamate. Okay, thank you. Some more questions for Anne. And then I can ask more. For the sugar, you say that sugar purification is very difficult and it take a long time. And I wonder how is it easy to preserve the sugar? Is it easy to get degraded like? Well, in case it was difficult to purify in this case because they were small sugar. And when you have very small molecules they're difficult to separate one from each other. In this case, it was lactose, it's disaccharide on fucose. So you need colon that are not classical, commercial colon, classical resin. So that was a difficulty, but then we can do it. Then if you have a monosaccharide, like fucose, no, it's not difficult to preserve. You just lyophilize and once it's lyophilized you can keep it easier to preserve than protein. If you have oligosaccharide, like blood group oligosaccharide that you buy from a commercial source, yeah, you better lyophilize them or freeze them and to avoid bacteria to grow on them. You should not make a bacterial culture or family culture in the lab, but yeah, just to be a little bit careful, not to keep them in solution on the bench, but they are not difficult to... It would be a problem if you try to make a drug for oligosaccharide and eat it or inject it because then you have the enzyme that degrades oligosaccharide. So when you think about glyco derived drug, you have to think about the stability in this case, yes. Okay, and the last question, you say that you also see the hydrogen bond. Is it being in the structure factor? Because we have interaction of water and then protein and then this sugar. So is it see by the interaction between them from the neutral? I did not get your question about hydrogen bond, if they... Because we learned like there is an equation and then there are form factor, structure factor. And so if we see from the I and Q and then we know what kind of... And different kind of information. So I wonder if we want to know the interaction between the H and O and basically hydrogen bond, is it seen in the structure mode? Structure factor or... Structure factor. Because we can also see the size, right? From neutral scattering. Yeah, basically my question is that how you analyze the data to get this kind of hydrogen bond? Yeah, it's not well represented here maybe, but you can really see the peak of the deuterium. So from the peak of deuterium and it has a continuity with the oxygen that bear it. So you can see how the hydrogen bond is going. At the moment what we try to see, if it's in this case where this very strong hydrogen bond, if it's not even start to delocalize and to be some hydrogen that is almost shared between the two oxygen. But normally this sugar, they have OH and from the density of the neutron and from the peak of deuterium, we can see direction of the hydrogen bond. Okay, thank you. So any more questions? Please take the opportunity. Victoria, can you move closer? Yeah, move closer to the mic. It's weak. It's weak. It's weak. It sometimes happens with my computer. Okay, move as close as you can. Yeah, it's okay. Okay, maybe like this is better. Yeah, better. Okay, I wanted to know why is the human milk is so important? What is the main component there? Maybe I didn't understand. Yeah, so in human milk, in cow milk, you have mostly, you have proteins that are important for the baby and mostly lactose. In human milk, you have the proteins and you have lactose and you have 400 different human milk oligosaccharide that are derivative of lactose. But with fucos, I did fucosillactose, sciallilactose, acid sciallic also. And you have all these complex sugars. They have two effects. The most important seems to promote the development of the good bacteria in the gut of the baby. So it's microbiome stuff, you know, at the moment. It's probiotics sugar. It helps the development of bacteria that are beneficial for the baby. And their second effect is to prevent from infection because if the baby have psodomonas or another Campylobacter infection, the bacteria will attach to this soluble milk oligosaccharide and it will not attach to the baby gut epithelial cell. So it's quite special for human.