 So thanks again. So my name is Frank Abel. I'm a physicist by training. I come from Germany originally, but I came to France in a university exchange with our partner university in Grenoble. So Grenoble is down here. It's at the height of Northern Italy. Often people forget this. We are south of Venice, for example, so it's amazing. So it's a nice place. We have mountains around it. And of course, from a scientific point of view, it's also great because you are here on the EPN campus, the European Photon and Neutron campus. So which groups together some nice institutions. So if you have the CIBB, we have the EMBL, we have the Synchrotron ISRAEF, and of course, what I will be talking about today, which is Neutron. So the ILL, the very historically one of the earliest Neutron centers in Europe and the first high flux Neutron reactor here and which was really very important to do biological experiments. So I'm myself working at the IBS, the Institute for Structural Biology or Institut de Biologie Structural in French, which is a new building now we were outside the campus we moved there, some little bit less than 10 years ago in this nice building which is really close to the entrance here and in walking distance to all these facilities. So I learned that there's a second lecture on small-angle Neutron scattering. So I will therefore limit my introduction to the technique to 10-15 minutes at the beginning and Susanna Teixeira will go in depth in the technique itself. But I will be necessary that they introduce some basic concepts of small-angle Neutron scattering so that you understand what I'll be talking about. So the idea is like if you short introduction to small-angle Neutron scattering. And then I show you two practical examples from the domain of protein degradation. So two practical examples where we apply the small-angle Neutron scattering and you learn tomorrow more on the theory and on the practical things like to fit data and so on with Susanna. So the brief introduction to small-angle Neutron scattering. So this is a structural biology technique, you know there are a couple of them so biological objects in principle you can be interested in understanding the structure and function at different hierarchical levels. So this is from a biology textbook. You have of course the level of the organism, the level then of smaller details. Here you go down by a factor of 10. In length scales you hear the fingerprints and it's of course with the usual, your natural senses like the eyes, you cannot go down in this scale very low. So for example here you would be able to see of course your finger, your fingerprints, the ribbons and so on, but your resolution of your eye stops somewhere, a factor of 10 above what would allow you to see actually the cells in your body. So you see this is the level of the cells then, then you have the level of the organelles, here's the mitochondrion, then you come to the macromolecules, here's the ribosomes, it's a single ribosome and then of course the auditomic level. So historically people were able to go down and see more details. So there's a light microscope that is invented then later of course NMR, crystallography, electron microscopy which joined now this club of techniques, restricted number of techniques which allow you to go to atomic details. So electron microscopy in favorable cases, NMR in favorable cases crystallography, when you can crystallize things, so this allows you to go to atomic resolution, allows you to see where the atoms sit in each macromolecule. The small angle Newton scattering is somewhere in between so you see from a scale from maybe 10 angstroms to about 1000 angstroms. So it's very nicely situated to see to resolve the shape of macromolecules. Unfortunately, it will not allow you to go to atomic details so you cannot obtain an atomic structure by small angle scattering alone, but it allows you to probe certain models of your macromolecules in solution. So just a few basics on the techniques as Susanna will elaborate on this. This is a scattering technique so you have some incoming radiation, it can be x-rays or in our case today neutrons, which are described as a plane wave. They will interact with the matter. So in the case of x-rays will interact with the electrons of your atoms in the case of neutrons, it will interact with the nuclei. So with the nuclei of the of your atoms, which means the protons, neutrons and so on. Since these objects, the nuclei are small with respect to the wavelengths. So the wavelengths is a couple of angstroms. The nuclei you know the size of an atom is roughly of a small atom like hydrogen is about an angstrom. But the nucleus is about a thousand times, 10,000 times smaller. So it's much smaller. So you can consider as a point like obstacle that is in the way. And this means of course that all the waves are scattered from your nuclei are going out in a spherical way. So the idea is that you can do it in two ways. You can imagine it in a mathematical way so you can for the physicist among you. So this you give a weight. This is a scattering length to each atom or to each nucleus, which messes the strength with which it interacts with the radiation. And then you have to take into account the face and so on with which it is scattered and then you have to sum up these waves. So there are some will be in phase so they will add up as a function of where you are on the detector others will be out of phase they will have a loss of signal. So if you go into details, Susanna will will will elaborate on this just to say this wave phenomena is not only in the microscopic world but it's also in the microscopic world and you will see it here to give you some flavor of hopefully a nice summer holidays is a beach on the in Italy and you see here I mean ignore for the moment the the the tourists there crystallizing under their umbrellas and avoiding radiation damage from the sun. That focus here on the waves coming from the ocean so you have these boulders these rocks you have small openings and here the spherical wave so this is like a perturbation of the wave and there's a spherical wave going out and you see this nice pattern here on the beach so it happens also in real life and this is the idea. How you can describe all this in practice so the other way is not thinking mathematically but thinking graphically adding up these waves and seeing what is the signal. Now word on by the small angle scattering. The principle is the same as in crystallography by the sample state is different so the individual proteins in solution there's not a regular arrangement in the crystal lattice. So you have all the waves that are scattered from a single object a single protein for example, and then you have to count how these waves at up at a certain point on your detector. If the object is small, as you go away from the direct beam forward scattering direction as you go away a little bit. You see here the waves that will get out of phase very quickly so there will be destructive interference, the signal will get weaker very. It will be will get weaker slowly so you have to go quite a bit away sorry you have to go back quite a bit away from the direct beam, because the object is small, and the difference between the phases of the wave scattered is determined by this distance here okay so if you have a small object small with respect to the wavelength you have to go quite a bit away in order to have destructive interference so a small object. Meaning small with respect to the wavelength, which is a couple of angstroms for neutrons would mean the intensity is very strong in the forward scattering direction because all the waves are in phase, they add up the intensity. And then as you go away slowly from the direct the forward scattering direction the intensity of the scattering object will go down. If you have a bigger object a bigger protein with respect to wavelength let's say this is Lysosimed this is a big object like a ribosome you have big distances with respect to the wavelength. This means big distances, if you go already a little bit away from the direct beam it means these scattered waves at points different positions within the object they get out of phase very quickly. This means as you go away from the direct beam the scattering intensity will drop very quickly. So the curve number two here down is from a bigger object. And this means this is so called reciprocal relationship between real space really the size of the object distances in object and the reciprocal space what you will observe on a detector okay. So this means a very small object has a scattering curve that goes to higher angles a big object has a scattering curve that comes down very quickly. You can make this to bring this to extremes if you're a mathematician you see this is a Fourier transform of the object in three dimension. This means a point like object, a point like object the Fourier transform of a point like object is a constant. So if your object is an atom or something much smaller than the wavelength, the scattering of this object will be a just on the on the detector flat line. On the other hand, if your object becomes bigger and bigger microscopic sky size, it's so big that it's for the neutron it's infinite infinite object. The Fourier transform of an infinite flat object is a delta function or point like function so all the scattering will go in the forward scattering direction these are the two extremes. And then you can describe this mathematically. I leave this to Susanna stock. So you can assume the conditions. You can measure your preferred biological system it can be proteins RNA DNA lipids and so on also soft matter of course colloids and things like that are used for for small angles getting, but you need a couple of milligrams per millimeter concentration wise, and then the volume typically we have here at I L on D 22 would be of the order of 200 micro liters. So let's say a couple of hundred micro liters of volume, and the concentration of your system, a couple of milligrams per milliliter. The sample state is important. It's not the crystal. It means normally your macromolecules are oriented. The orientation is arbitrary, assuming that orientation of one macromolecule doesn't influence the orientation of another macromolecule. Okay, this means you have an isotropic arrangement in all different orientations possible of your macromolecule solution and this isotropic orientation leads to an isotropic pattern on your on your on your screen or on your detector. So this means this point of symmetry is the direct beam here and then as you go away in different directions. You have the same average loss of course there are statistical fluctuations on how the Newton's actually scattered but on average, you have the same loss if you go up here down to the left to the right. As you go away from direct beam the loss will be the same on average. If you lose information, if you sum up here concentric rings of intensity, you just define a distance from the direct beam a scattering angle, or wave factor transfer, and then you have an intensity that decreases as a one dimensional curve, and that's the information you get from small angle scattering it will be in general for isotropic systems a one dimensional curve intensity as a function of angle. Now as I said, when you had very small angles means in real space you had a bigger distances. This means at very small angles you have some average information on the whole integrating on the whole macro molecule. These are global properties, you will get be able to get the molecular weight of your macro molecule and you will be able to get something that's called a radius of duration, which is a measure of the distribution of your atoms in your system. The radius of duration will be big. If the system is stretched in something elongated it will be small if it's very compact like a sphere like a structure. Now we can measure roughly everything here from single amino acids, if you want to get radius of duration of a single amino acids it's possible up to very big objects the biggest objects, macro molecule objects like viruses ribosomes and so in the order of the range. Now, the globally information provided by small and scan is all the American state of my chemicals, the shape of the molecules is the, does it look like a banana like an apple does it look like a donut like a potato. If you have an interaction between different macro molecules you could titrate a partner or ligand and see how the mass of the object what is the stoichiometry what is the conformational change if you have partners interacting. And the nice thing of course is you work in solution so we'll play around with your solvent conditions, you can work with pH salt, ligands temperature pressure and so on so that's a nice thing you can look how your system reacts under these different conditions. Now the important point for neutrons, more widely used in neutrons and for x rays is so called contrast variation which allows you in more complex system composed of several partners to focus on the signal of individual partners. Now globally of course you can go do more than just the molecular mass or the radius of duration there's also modeling I won't go into too much detail I will show you practical examples later. And sans do with respect to suck so remember x ray sucks observes electron density. The problem is that the electron density of the different objects in micro molecules are not too different and another problem in, you cannot work influence a lot the solvent density of for for x rays so the water solvent density the electron density of the electrons is more things in neutrons. So what do I mean by this. Imagine you do some modeling of a large complex composed of several proteins blue the red, the green the yellow one. You will get something like an envelope for this looks like low resolution electron microscopy you will get some envelope of this. But the problem will then be, where do the individual partners sit within the envelope okay is the green protein really here or is it sitting here and so on. So if x rays, you wouldn't be able to distinguish this, because, on average, the electron density of all proteins, even though that the sequence might be different. It's, it's very similar, and you cannot distinguish them so by x rays you can distinguish globally the signal from one protein at low resolution from the other. And you can this by changing the interaction with the neutrons and how do you do is you have to change the nuclei, because neutrons they interact with the nuclei of the of the of the proteins. This means you have to do deterioration so this is the idea of contrast variation years and example from some women from a tribe in Africa with the traditional traditional clothes and the in front of this background so getting invisible in a certain way. And you can calculate this, I won't go into details just saying here. So son, I will take more time to explain this. So by changing now the solvent properties scattering properties for neutrons by changing normal water H2O against heavy water which is D2O with deuterium instead of hydrogen. So you can change the properties how neutrons interactive them because neutrons. You can imagine that a neutron index different be for single proton which would be hydrogen atom or with a deuterium, which is a hydrogen plus a plus a neutron in the nucleus. Okay, so there is principally a difference in how it interacts. So you can then trace these things you have a certain baseline here for the water going from normal water H2O to heavy water D2O. So the scattering properties of individual macromolecules here for neutron for proteins for RNA and so on. And the nice thing is that they are different. So our nays get us differently with neutrons because it's chemical composition is different and especially the density of hydrogen atoms. And then again, the scattering properties for limits are different so you have the possibility then to to distinguish these different partners, they are then special points here where the water baseline crosses the protein. This means here is something called the scattering length density. So if the scattering length density of the solvent is the same as that of the protein means the neutron can no longer distinguish between the scattering that happens in the solvent and then happens in the macromolecules. This means the protein becomes invisible and this happens around 40% D2O in the in the solvent so at 40% roughly D2O in the solvent depends on the protein sequence. The electron will become invisible. And this means then you, you can focus on other parts of the system for example. Now here just graphic and analog on from refraction so this is refraction from light. You have a plexiglass rod you would see it here in the air and underwater. You add for example glycerol which changes the refractive index of the solvent. At some stage, you see the plexiglass rod it becomes invisible underwater. Now, would it be possible with x-rays so here is the situation of x-rays on top of it so normally you would find in literature values like electrons per angstrom cube which is an electron density that characterizes the system. And then I express it in scattering density which is again a measure of how strongly the x-rays interactive the system. And you see of course also electron density you know RNA has this phosphate backbone and so on so it's much richer in electron density that proteins. So there's different level that's all right. The problem is that you can no longer make all the partners invisible by x-rays because you would have to change the electron density would have to reach the same electron density as a protein in water, which is lower. So it's almost impossible to reach an electron density as high as that of RNA. So there's no possibility nowadays with x-rays to do this contrast variation as you can do it with neutrons and see here. Neutrons going from H2O D2O, you can cover all the range of these interesting molecules RNA DNA protein detergent lipid sense on. So that's how this would work you imagine you have a protein RNA complex. As I said RNA has as a bigger contrast than a protein here at 0% D2O, you would have a black object representing the RNA and if you have to protein partners in the complex they would be grey objects in front of a white background at 100% H2O normal water. If you go into around 40 42% D2O the protein would become invisible and you would only see the RNA. This means really you see the RNA as this bound in the complex so it's not free RNA that's important so you could by comparing this image here with this image you could see if there are some differences between this if you measure the RNA alone, or if you measure it in the presence of the protein that's important. It's the assemble complex and then if you go to a higher degree of D2O around 70% D2O depending again on the RNA composition the RNA here becomes invisible, and you see only a signal from the protein. So what I was saying at the beginning with x-rays you can distinguish between two proteins in principle with Newtons you can't either, because the chemical composition is the same for normal proteins, but you can do deuteration and so I will duration you will hear about this so this is exchanging the hydrogen atoms by deuterium atoms in your in your protein so this is chemically you grow bacteria and media so we'll hear about this I guess later in the course. And then you can distinguish one protein from another imagine you have a deuterated protein a hydrogenate protein you reassemble them in the complex. You can mask the signal of the hydrogenated protein at around 40% D2O, you will still see the deuterated protein, then you can go to a certain point at 100% D2O, where you come close to the matching point of a deuterated protein. You can distinguish it really because the deuterated protein has a stronger as a bigger scattering density, but there are special protocols you will also I guess hear about, which allow you to adjust the level of deterioration of your protein in such a way that it comes down a little bit let's say 75%. And then you bring this line down. This means you can then mask the signal of this partially deuterated protein in 100% D2O. And then you can look on the hydrogenated path. I jumped this so we have spent 15-20 minutes with theory and now I come to these practical examples. And again there will be handouts of my lectures and we can also discuss this later or interrupt me if you like. So I showed two practical examples now on systems that are started in our group as Bruno von Zettis' Elma group, extremophiles and large molecular assemblies here at IBS. And so I showed two examples, one static example where the system is not changing with time and then the time-resolved sun studies. And the question is of course with respect to other techniques, what is the unique insight that these neutrons or small-angle neutrons scattering can provide for these systems. So now what is this protein degradation in cells? What's the general topic? Now you have different cells in your organism or even single cellular organisms. They are not static over time. Of course their composition, of course their morphology changes over time, but also the content in certain protein species as a function of where they are in their life cycle. So if there's growing, dividing, if there's external stress, temperature change, nutrients, nutrients are coming and so on, or if you have at the early stage in your organism developments or cell differentiation, will your cell become a muscle cell, will it turn into a brain cell and so on. So the global content in proteins and the structures of course is varying a lot. So this protein that is the ensemble of proteins in the cell needs to be produced, it needs to be controlled, the quality, and at some stage if it's longer needed or if there are some other things happening, it needs to be destroyed in a controlled way, okay, and recycled. So of course there are several levels of control, you have gene expression, you may notice if you come from biology of course how proteins are produced specifically and so on are the activated production, but then also you need specific degradation and I'll talk more about this. And of course you can imagine if these things go wrong, they're all kind of bad things happening, tumors, neurodegenerative diseases, aggregation, fibrillation and so on, aging of course also which is what's not a disease, it happens to everyone, but it might happen in an uncontrolled way. Now here, a general image overview of this, so you have all these regulatory processes going on at production of proteins then also the quality control, if there are some slight misfolding there are some kind of repairing mechanisms available in the cells or about the chaperone systems and so on so they will then unfold the proteins in a certain way, but then if a protein needs to be specifically destroyed, there also is miscellaneous, so the first major player of this is the proteasome system, it recognizes specifically the proteins to be destroyed, they are labeled in the case of eukaryotes, you may have heard about this ubiquity system and all for lower organisms like archaea mechanisms, archaeal mechanisms and bacterial mechanisms, there are small so-called degrons, small sequences in your proteins that the proteasome recognizes specifically and then attracts it to it and then drags it into this proteolytic chamber to degrade them. What comes out are smaller oligopeptide pieces of sequences and then there's a second class of systems like they're called the peptidases, they take into a charge the smaller oligopeptides they can be of the order of 10, 15 acids in length and then cutting it down into smaller pieces, so my first talk will be a static sun study on this lower part of the system on the so-called TET system and the second part of my talk will be a time-resolved study on the upper part of the proteasome system. Now, the first example, so we're working with archaeal systems in our group, archaea are the third kingdom of life, so they're not bacteria, so they are bacteria, archaea and then eukaryotes, okay. So, and these are from extremophilic organisms, it's in this case pyrochococcus oricoshi, which is small beast like this, it lives close in the down in the bottom of the oceans close to this hydrothermal vents. So it's a very extreme environment, high pressure, high temperature. So these are very robust systems, I mean, of course, there's a global interest in on how these organisms involved in the context of the origin of life, but I'm not focusing on this today so it's more on the biophysical properties for the structure biology community often they work with these proteins or some molecular systems from the extremophilic organisms because they have a certain number of advantages for structure biology, they are robust as I said, stable at high temperature and so on. So this is this TET system, so this is one of these peptidide systems that degrades these pieces of oligopeptides into smaller single amino acids, okay, it looks like it's a dodechimeric edifice it has some openings here where the peptides will enter and then go out again. And the catalytic site is buried in the in the middle, there are several catalytic sites that are buried in the middle. So it's a it's a big system it's 500 kilodolts was discovered in about 20 years ago years ago. And it's the they come in different flavor, these are monomeric building blocks with certain specificities towards certain amino acid residues okay. And then there's a so called TET2 system which is more sensitive to cleaver neutral amino acids, and then there's a TET3 system which more evolved to cleave basic amino acids. So my colleagues by doing biochemistry they found out a strange thing so the the catalytic site I should say is here on the apex so you have it here in the large of you there are three catalytic sites on each apex. Okay, it comes in and then it's cleaved here. So my colleagues they found out some some some strange thing. They were mixing homododicomeric TET2 particles with homododicomeric TET3 particles in the solution. And then they were also assembling a certain kind of hybrid systems or heterododicomeric systems, but in the same stoichiometry so in the solution you have the same concentration and same stoichiometry of TET1 sub units and TET2 sub units. They are just assembled in a different way. So here's a mixture of two of homododicomeric particles and here mixture of hybrid particles okay. So what they found out is that the efficiency of the system is increased. If you have these hetero polygomeric particles. So the question is of then of course how why is a heterododicomeric particle at the same stoichiometry of the catalytic sites and so on, more efficient than this mixture of homododicomeric particles. And what do these heterododicomeric particles in the solution look like. So if you study this, you, you might imagine we use suns and contrast variation. So the idea is now we would deuterate one of the partners, in our case TET2, and let the other one that had 31 hydrogenated, which would allow us then to mask the units from the test three at about 42% D2O and having applied this match out labeling so that the particle, the deuterated particle becomes invisible 100% D2O which was developed at the, at the D lab at ILL so there's some reference here. You would be able to match everything deuterated at 100% D2O. Now what is the idea now. My colleagues were constructing assembling this heterododicomeric particles in solution. You can imagine there are different topologies we knew that the building plot is a homodimer, so it's on the crest here of this tetrahedral particle, but there are several different possibilities you can think of. So you could have a single dimer deuterated in the particle you could have two dimers, you could have three dimers deuterated but they can come in in different topologies. The internal techniques, even mass spec it's very difficult to distinguish these cases of course the mass is the same of this object you have six deuterated partners and six hydrogenated one and here the same, but the internal topology looks different. You can't distinguish it by sucks because the TET2 and TET3 are very similar so it's very, it's very tricky. So in this case you could think of this topology you measured in solution you would measure it in small small and newton scattering you would measure it 42% D2O to mask the signal of the hydrogenate partner look at the structure of the deuterated partner and then do the inverse measure the 100% D2O and look at the hydrogenate partner not seeing the deuterated partner. So we are purifying this and then measuring in solution. So all the possible topologies you can think of apart from of course the homo-dodecameric two extremes which I do not show here. And then what do actually the scattering curves look like. So if you measure these different objects you can calculate it for these topologies if you do an in silico reconstruction of your heterododecamers and you can fit it with programs against experimental data. In some cases of these topologies the fit is good in others is medium so what I call yellow is maybe maybe and here green is a good fit in terms of a chi-square value so it says how well the theoretical curves agrees with your experimental curve. But it's not enough as you see to distinguish which one is it actually could be this one this one this one. So you have to look at other data, you have to look at 42% 70% 100% so if you do it. You see this here. At 42% you mask everything that is light gray. In 100% you mask everything that is dark gray and in the 70% you're in between the two of them. And of course your system your actual system in solution it must be in agreement with all your experimental data so it's supposed to stay the same it's very robust particle. In 0% d2 in 0% d2 or in between the topology and the assembly of the particle stays the same. So the actual topology that you looking for is the one that must be in agreement with all your experimental data. And as you see here everything is green there's only one case possible. And this is called what we call this Z like particle because here the arrangement of these label diamonds is a set like structure. So we have a unique power here of sons to look into inside this complex particle and get the topology of the individual of the different to different partners. Now we have here a little bit more data more detailed fit here you see it's it's relatively nice here some noise level and maybe background from the direct team, but overall here this structure fits very well. The curve. Now we have also this POF function so Susanna will maybe introduces more detail this is a measure for the distances between different atoms in the system and here. There's something very weird if you hear about this later in your in your in your PhD so if you suck state anomaly the POF function of also positive values because all the electron density is in your protein is above the one of the two and but here we have the case where some of the in the 70% data, you have some of the deuterated, you have the deuterated particles that have positive contrast, the hydrogenated ones have negative contrast you have your very weird and funny, and funny and that they're very rich on the information you get on the internal structure. Now, going further we have now also complemented the study with cryo M. So we have detected by cryo M. There are certain intermediate structures open structures which are not going to make structures but hexametic structures in some kind of freeze in the in the assembly during the assembly mode so we see that there are certain intermediate forms before the full assembly of the complex. And the idea is we use this information combined with the small angle Newton scattering so my student Alex had prepared a small movie here I hope this. You can see the movie so there's the building block. It's a homo dimer can be either tattoo tattoo three. It comes together with other homo dimers forming either a closed intermediate hexametic structure on open intermediate hexametic structure. So normally the two units should move but but my student did the simplified way so there should be some some symmetry here. So this hexametic intermediate structure that it was this cryo M structure here. And that would be the second intermediate hexametic structure coming from the complimentary tech particles or either tattoo or tattoo would come together, and they would form now this double z like structure in the fully assembled. final model. Okay, so the idea is you can then with this intermediate information on the assembly pathway and the final structure that is validated nicely by the suns, this heterodicameric double set structure. You can then speculate on the assembly pathway and you can exclude certain hypothesis of the assembly which would end up with this kind of topology which we did not observe. So this data so only this one is valid. So the nice thing is also that in this very special structure compared to these two other possibilities. You see each catalytic pocket which brings together, which is composed as a hetero catalytic body. This means you have one catalytic site here from the light gray particle. So the catalytic sites from the dark gray particles or tattoo tattoo three, they're complimentary remember the first slide one is more against neutral the other against basic amino acid residues and cleaving. So you would assemble in each catalytic pocket, a hetero catalytic specificity so we group them together locally complimentary catalytic sites and this we believe is the reason why this particle here. The hetero particle assembled is more efficient in cleaving these longer oligo peptide substrates in the end so this is a combined suns em and crystallography study because we had the single building blocks, which we combined in silicone for the sun study from crystallography. So that was the first part of my lecture static sun structure, which allows you to look inside a complex assembled multi protein complex. The second part is time resolved suns and it's about this upstream, what's happening upstream your own we are now dealing we were dealing with these oligo peptides but how do you get these oligo peptides. The oligo peptides you get from the proteasome particle. Proteasome is a very big object itself the order of two megadolm for you periods. And it's really big so you have this ubiquitin labeling to the polypeptide chains here in the proteins that are here in dark red. And over the past three, four, five years with the cryomers revolution, you have enormous sophisticated models coming out of this so there was enormous progress in the understanding of the function of all this. And even people were able to, to, to get atomic resolution models of the block substrates or not only of the proteasome alone but also in the presence of the substance or see here this. And as it is being unfolded here so the upper part is the so called them. How to say the regulatory particle this is the one that recognizes the proteins to be degraded it tears them tears on them so it unfolds them. And then it brings it down into the catalytic chamber here of the catalytic core particle okay. So there's a new carrier says ubiquitin labeling and in other organisms like bacteria and and are here. They're just sequences on this or surface motives of the proteins to be degraded. Okay. And so you have always a regular deep article that recognizes specifically brought to be degraded because you don't want all proteins and specifically of course to be degraded. And then it guides them directly into prodigy. So crime has undergone this revolution and they are over the last two, three, four years the enormous high quality structures the point is of course the limitations kind of static snapshots. So you see certain steps of this unfolding process, but you don't get a global picture of the overall process. So, now for some studies this eukaryotic particle is too complex there are dozens of different proteins it's very fragile so assemble and you need a homogenic population and so on of course in crime you can select families and so on you cannot do this in the sense you see everything that is in solution. So the assembly the labeling of this is enormously complex so we're working with a more easier version of this. The easier version comes again from these are keel extremophilic systems now from a ton of color color coconut she it's no longer pyrococcus. It's again an organism that leaves the lips leaves deep down deep down in the ocean at very high temperature again it's a very robust component particle and also very recently here now. In the studies you were also first cryom structures coming out at some kind of intermediate resolution from this particle. It's easier than the eukaryotic particle because here the pun so called pun particle here which is a regularity particle in this case it's a single species of amino acids is a hexamere, but it's always the same copy of the protein, whereas here in the eukaryotic, you have 10s of different proteins here. And for the core particle which has also 10s of particles different proteins here you have only two species that assemble here so already from the composition it's easier, but also from from the topology it's easier. And the big advantage again for our son study is that you can manipulate this so you can, it's active at higher temperatures. It doesn't work at low temperatures so the idea is now for the son study. You can have the inactive particle at 40 degrees Celsius, and then you go to higher temperatures, and you can activate the function so you can use temperature as a, as a trigger for the reaction. I see there is a question in the chat I'm not sure if I'm. Okay, no this is a general remark okay so there's no specific question, but feel free to interrupt. Let me move on. So, again, you have some kind of static structures yet very good resolution for crime but the dynamic process how do you start it. So again, small angle Newton certain comes into that. So, well, maybe a few remarks here again to to how the process goes so these are specific motives here. Here's a review from a famous group in the US, you have conformational change unfolding sub unity station you have certain surface motives that are recognized then by the pun, which tears it which draws it by an ATP driven mechanism down so you need some energy to unfold by unique ATP. It draws it down into this catalytic chamber. So the idea is we would like to do a time resolved suns. And if you remember of course. So we would like to see this process we need a substrate and the good substrate that has been validated by other groups and it's nice because it's fluorescent is green fluorescent protein GFP, because you can not kind of only study the structure of the protein but if you do fluorescence in parallel, you also know something about the folding state of the protein so our subset was a certain labeled GFP there's a small tag on it that has been validated as a recognition motive for the pun so you can draw a can you drag down a part of the of the GFP across this channel into the catalytic chamber so we would like to study this system in a time resolved sunsway so we'd like to follow what's happening to the substrate of a course of time, but also if there are conformational changes here for example in the pun molecule over the course of time of unfolding. In the first part of the project we are doing the simplified version only substrate GFP and only the unfolders pun. Okay. So this was a study from my joint ILL, IBS PhD students at Ibrahim. And the idea is the following one. You want to look on the one hand on the substrate on the other you would like to watch what is going on with punk during the reaction. And you're labeling again because two are both are proteins you cannot distinguish them if they are unlabeled. So the idea was to have one experiment with deuterated GFP and the other with hydrogenated pun and the other one with hydrogenated GFP and deuterated pun. So you have to activate the reaction by putting the sample at 55 to 60 degrees Celsius and supplying ATP, and then you would like to follow what's going on here during the reaction. Now, for all these experiments very important to have sample characterization. Before you do use these precious samples labeled samples, you have to produce proteins you have to come up with the reaction mix you have to optimize your ATP and so on you have to characterize all this offline before you do the actual experiment and coming to a neutron center. Okay, so you do cell filtration experiments and you do bio core experiments to check the interaction you check what happens at different ATP and other experimental conditions when is the sample very mono dispersants on so you optimize all these things beforehand it's very important also. Often I recommend before going to a neutron center do first an extra experiment to characterize the sample you don't need labeling. So you, you learn already something what happens to system is there some dependence on concentration do the success experiments also in a normal way first Okay. So the second part now we want to look what's on the labeling so we are, we are watching here in this case we are putting ourselves at around 42% detail. We always mask the hydrogenated part of the system and we watch what's going on with the deteriorated part. In this case it was a perpetrated sample because we wanted to have a maximum signal over noise from the label partner we could have chosen the deterioration level a little bit lower it's less expensive. So the signal is always proportional to the square between this distance here between the two scattering length density so you're make this, you do divide this by to the difference is getting length, you divide the signal by four. Okay, so please it's important something to be aware of. So of course we had to check that once the hydrogenate partner is really masked at the digital level of the soul we chose. So that's why we compared here deteriorated pattern in the presence of hydrogenated GFP and it's so you see the two scattering curves are the same so the hydrogenated GFP does not contribute. You can also look at this individually here's the hydrogenated GFP and the buffer level, you have no signal, the same, the other way around now hydrogenated pan disappears in this 42% D2O buffer, and again the two scattering curves, if you combine deteriorated GFP and deteriorated GFP and hydrogenate pan is masked. So this is again a quality control and it's amazing, because the GFP is about 28 kilodolts in size, and the pan system is more than 10 times bigger than this so a single pan particle again the the signal that the particle gives. It's goes with the square of the molecular mass so it's very sensitive to big particles, small ingredients. So these are all quality controls before the actual experiment. So how is the actual experiment done. So the system is kept at ice at 40 degrees Celsius, and then it is put on into a sample rack that is termostated at 55 degrees Celsius. If you put this in there, it will trigger the reaction. Remember, this is an extremophilic system, thermophilic system means at 40 degrees Celsius the activity is very low, but at 55 degrees Celsius, it really turns quickly. So if you put this in, you run out of the of the hatch and then you, you switch on the neutrons, and you measure what's going on. So you have two windows here this is special sample cells or it's not the usual standard sample cell that is used for nutrient scattering and small angles so this is the 22 instrumented isle. And it's our contact our collaborator on my tell who developed the system here so it's a joint device that allows you both to measure the scattering of the new turns of your system. So there's a second window when you put the, the, the quad cell where with the sample in it, which allows you to follow the fluorescence. Okay, so there's UV light you you excite the system and then you, you, you look at what's coming, what's coming back as fluorescent signal. And you measure this in parallel. So what comes out in the experiment. And then fit the signal and you, you, you get an information you get a time scale for the disappearance here of the native GFP signal. You have a disappearance of the fluorescence and then exponential decay, and you have a buildup of aggregates. So if you look at the different curves at the very beginning so this is 45 seconds because we have a 15 seconds delay to run out of the hatch, this is not yet fully automated with robots and so on so first try. This is a 30 second exposure time so this is a signal after 40, 45 seconds the red curve corresponding to this one. It's pretty close to the, to the natively folded GFP so you can fit it again with some programs. And you see it at very beginning of the reaction the GFP is native native state, but then, as you follow over time so all 30 second frames exposure frames, and then going through the action up to 50 minutes which was the time we had per sample. You see that the protein unfolds and aggregates. Okay, so the mass of the object increases you can see this by the change in I zero intensity but also the, the shape of the curve changes over time and then you can reconstruct this low resolution model. So I think that the GFP is being unfolded but then being unfolded in the longer refolds correctly. And then there may be hydrophobic patches maybe at the surface and then it will will aggregate in this pearl like chains. So we can characterize with this over time what is the rate of direction how strong and quickly it aggregates so the important thing is, you may have that you turn scattering is low but this higher flux instrument. You can go down to 30 second exposure times and getting still very decent data sets that allow to do some modeling. Now, we see that the GFP aggregates with a certain rate. Okay, this is not surprising we have only unfold us we don't have the other partner. So this is the pattern system. The pattern system over time again you have here the initial state at around 45 seconds and intermediate state around five minutes, and then the state at the end of direction 50 minutes. And you see the, the sun's curves there are slightly different so the I zero intensity is the same this means the object keeps its oligomeric state, but the slope at the beginning changes. So this is related to the rate to the rates of duration as you will learn with Susanna. So it means at the beginning of direction the, the, the particles in the resting state in a relaxed state then during the action while it unfolds the GFP it will contract so there are some conformational motions, and at the very end of direction when the ATP is consumed and the GFP is completely unfolded. It goes back to its resting state. So you plot the radius of duration, which is a measure of the extension of fun particle over time. You start at the resting state about around 66 angstroms, a registration you go down to a contracted version which is the working complex at 60 angstroms and then it relaxes back. Once the ATP is consumed and GFP is unfolded to the initial resting state. So together in the first publication on this limited part of the system, we were able to come up with a certain models you have the initial setup of the, of the sample you have the pan particle he symbolized you have the fluorescent GFP particles. So that it will, it will then be transferred across the channel of the pan it will unfold, it may, maybe refold partially but not completely and be then reaction prone so over time, there will these GFP aggregates that build up. So that's the first project now we come of course to the question what happens if you add the actual if you have the hollow complex, as it works in in in an organism okay. So you have both the unfolders the pan complex, but you have also now the peptidus core particle which takes into charge these unfolded chains of the substrate and cuts it into these oligomeric pieces. So we have a second PhD thesis from Emily, which was my student, two years ago at at IBS and we also did again the experiments at ILL 22 on the same device at show. So here again, you have now the information structure information you have to of course you need somewhere also a reference what happens to GFP alone at 55 degrees Celsius, which is a relatively elevated temperature, you have to be sure that GFP doesn't unfold. So this is why we have also all this reference curve is here at the neutrality GFP alone at 55 degrees Celsius during 45 minutes. You see essentially nothing happens that's reassuring because it means GFP alone in solution doesn't unfold spontaneously everything that happening is really the consequence of the presence of the complex. Now this is what essentially what I showed before this is just a control experiment, the GFP hydrogenated pan you don't see the pan signal what you see here is really what actually happens to GFP during the reaction. You see again this is a log log plot now before it was a log linear plot so it looks a little bit different but it's the same message it unfolds over time. Okay, and aggregates over time. The interesting part is here. Okay, so you have here, what's going on you have both pun and 20s particle this core particle here present okay it's called 20s comes from sedimentation coefficient. Now what happens to the signal over time. You see the signal goes down, but there is no change in slope not a significant change in slope. There is no elongation, no increase in intensity it's a decrease in intensity. So the signal of the GFP part becomes weaker. What does it mean. It becomes weaker this means part of the population of GFP disappears. And then the second feature here is that higher angles. So the red curve you see also the, the, the data at higher angle red curve here is here and the black curve it goes. At higher angles but it comes up at high angles. Now what does it mean. It goes down at lower angles means that GFP, the native defaulted particle disappears. The fact that it comes up at high angles means remember the reciprocal relationship when you look at high angles it means it's something small in real space. This means if the signal increases at at high angles here means that something small is appearing during the reaction. This means if you have the hollow complex GFP disappears over time the native GFP. There's no formation of aggregates which is very important, because imagine during this process of destruction of proteins you would have the formation of aggregates in yourselves it would be disastrous. And you have small objects that appear, of course the small objects are nothing else but the polypeptides the oligopeptides that is appear here so I go in detail in this now. In addition to, if you want to model this now over time you need some idea what are these, these, these small object, the products of direction and you do some mass spec. So you have some kind of distribution of lengths of these oligopeptides it's not a single species several species of the order of a couple of kilodaltons to maybe something like a 10 may or 15 may okay. You can also inspect on a macroscopic level what you saw now on the individual macromolecular level you can look at a macroscopic levels you have to look at the actual samples you see here these are these quarts give us. You have the new term window here you have the UV illumination here. This is isolated GFP it's a green solution well it's green fluorescent protein that's how it should look like. And this is the intermediate thing so you have the DGFP and only pan unfolding remember aggregates aggregates in the sun sample they look like trouble the like milky opaque solutions that's exactly what's what's happening here so you have this milky solution this is, you see the aggregates already by the light scattering. In the case of you have DGFP H pun and 20 s the whole system. Well, there are no aggregates that's good, and the green fluorescence goes down this means there's less green fluorescent protein that's exactly what we saw. So on the macroscopic level, you, you have an agreement of what you see by eye to what you interpret at the macromolecular level. You have additional controls that the GFP really disappears with faster blood sense on I won't go into details but you will have a handout of the lecture and if you like and you're interested you can also read the publication. Now how do we proceed now we want to fit this data over time so we have a native population of GFP we have substrates. We might have an intermediate state something that is being in the course of being dragged so the proteins on on on how do you call this in English unspooled I think so you have something you tell you you unfold it. Now we can fit this you can try to fit it with two populations to scattering curves over time with this native structure which you know, and then you take some average unfolded intermediate. So the results from mass spec we chose a 10 male, you cannot fit a distribution of substrates it's too. It's too much information for the level of quality which you have in the 32nd exposure times of the neutron so there's a certain noise level as you may appreciate. If you look at these gray curves gray data sets, and having now a distribution of substance would be too much. We chose somehow an average substrate which we chose as a 10 male based on our mass spec data, and we fitted the population of a fully GFP and of this 10 male oligopeptide. And we have then certain populations of the program is called oligomer so it's. This is a sequence from the access package that meet respect once group at Hamburg which you may know. So this is then how we follow this as a function of time and of course before also the ATP but this, this will do offline. A second possibility is then you fall, you fit three species you fit GFP a substrate, sorry, a product, and some kind of intermediate structure which we constructed a little bit. And dirty from the cryo am so to have a rough idea on a part of the GFP that is still folded and the long unfolded thing. So what is the results of this, the results are here, okay. So you have your time axis, you take this unfolded intermediate from from a snapshot from cryo am and constructed so girls different substitute we somehow put the GFP and to get out so it's it's a construction just to have a rough idea on an intermediate would be that size and in that state. And then you fit the populations. So you see at the beginning there's a lot of folded GFP. It goes down to certain base level and then the substrate comes up. And here, the intermediate state is not so clear. So often it's zero sometimes it's five maximum 10% so if it is there. So even time during time point during the process, it must be very lowly populated. Okay, so it's a quick intermediate state. Now what else do you see combining all this information. If you have to be alone, you have to I see your intensity which stays the same. Over time is a reference sample. The Archie stays the same or time this is a slight loss of fluorescence which is due to the, to the high temperature but the globally the GFP remains the same. Here again, the Archie goes up very quickly to aggregates I zero goes up aggregates fluorescence goes down because the GFP is being unfolded. Next now the hollow complex GFP fluorescence goes down and very nicely hand in hand, the I zero intensity this means as GFP disappears the fluorescence disappears. And you see the eyes, the Archie is more less the same as a slight decrease because of course the Archie is a mixture of the Archie of the GFP but also the small products. So that's a summary of all what we measured. You have the specific time ratios for the I zero for the disappearance of the GFP which is measured by the, by the scattering curve by the I zero. You have a certain time rate which is roughly the same for the disappearance of the fluorescence for the falling state. You have a third time rate which you measure offline, but under same conditions as the sun experiments of the consumption of ATP. All three time rates are the same. And you have a fourth time rate which comes from this oligomeric fit, which is a little bit lower here. Okay. So this is a global picture. And you can come up with a global interpretation then of all this and I have one or two minutes to go and then then I'm done. So this is the mechanism we propose on this. You have a certain transit or interaction of the GFP substrate with the complex. At this stage you can still dissociate the nothing happens. No problem for the cell. But once you have this unfolding process engaged over point of no return. It's no longer possible that as dissociation. So we interpret this as the GFP being unfolded being dragged across the central channel but keeping the two partners the pun and the 20s together which is very important extremely important because if you had the liberation of of unfolded intermediate aggregation prone at immediate to be a catastrophe yourself. So we could show by the suns which is very extremely sensitive to aggregation that this doesn't happen the assembled process works very nicely. And the third message is that the unfolded intermediate must be very, very transient of the order of five a few percent maximum. So the unfolding process going through the channel intermediate state is extremely quick. So there are some offline characterizations very important again by a physical and so on and so on. Maybe last two slides. Could this have been measured by sucks. It's very important so x-rays. I was saying x-rays are sensitive to electron densities. So you can see for 20s pun and GFP are the same problem is pun and 20s particles together they are one mega dollar and the GFP is 28 kilo dollars. So you can calculate this in silico what this same mixture we had in Suns would have produced in x-rays. Okay. And the black curve would have been the average curve of all three partners in these given concentrations together. So what you will actually want to look at is the signal from the GFP signal from GFP is the screen curve and you see here of mathematician or physicist you know this logarithmic curve. These are two orders of magnitude. So the signal of the overall mixtures 100 where the signal of GFP is one. So it's completely would have been completely impossible by sucks to look at what's going on to the GFP in this complex mixture. The other way around it would have been the same if you want to look at pun in the presence of a very large aggregate here pun it's again more than all the magnitude of difference you could never have observed what's going on on the confirmation changes in pun. If you have at the same time the GFP aggregates forming in a sucks experiment and only do it with neutrons and this also again a paper for my for my student. So that's it my conclusions. Time Resolved Sons allows you nowadays and I hope the same will be the case at the ESS once it's completely operational to allow the sub minute range so we are hopefully also we did checks at five seconds, of course at one stage. If you go to one second two seconds the signal becomes so noisy that you no longer can fit a real form factor you may get a registration but you cannot longer fit models. So of course we'll see how far ESS will go I hope they will go very far in this time resolved experiments. I'm very optimistic about this. Now you can combine this also with online spectroscopy it's very important to complement in this complex biophysical systems with other techniques in addition to neutrons. The other side is dynamic process of important biological system is not only the pan system I'm showing there are a lot of biological systems that uses ATP other things, and have a time evolution. It's complementary to static techniques like cryo and crystallography also to solution like NMR. And of course for for your new expense to select and depth it biological system. And it's also extremely important to have a good deal up to have the the correct deterioration that you want in the system. This is thanks to all the people involved it's a collaboration mainly between I'll I'll I'll yes we had some funding from ANR so our local contact and collaborators and Mattel with all these nice sample devices she's developing and 22. Thank you very much for the D11 particular to Martin Muller who produced the samples and these are my students Zia Ibrahim Emily my you did the work. All these nice systems developed by by Bruno from said he which is a group leader in our group, and we had also the tech system here which was from another student from Alex up earlier before. Thank you very much for your attention. There's a question actually. So, you know, who says why high flux is needed for kinetic. Yeah high flux means you see you have a 28 kilodolton system. You have a few milligrams per milliter you have a solvent scattering. So the signal from the sun's from a sun's facility usually and unfortunately it's not the same as for for singleton singleton. So the shots we did in 30 seconds here you could have done them probably in 100 milliseconds or something like that. Problem is, as you said there's no, there's no, how to say resolution you cannot see what you want to see because you have no labeling in socks. And then a second point is also x-rays are destructive on your sounds if you do illuminate such a sample during seconds and seconds in the sex facility it would be impossible. So you need high flux neutrons. If you go I mean there was formerly facilities like LLB and so on which is very nice facility but I think this kind of time resolved study with 30 seconds of resolution would not have been possibly with a medium flux So high flux is important. You can do it at ILL. You may do it probably at FRM2 and things like that. I hope really that in the future you can do nice things like that at ESS also. So you need this high flux for this very big scattering biological systems. Neutrons are much more sensitive to metals and things like that. So if you have, if you study magnetism, material sciences and things like that, or you can go to systems with very high concentration. So here we have two mixed per mil. It's about a per mil of the volume of your sample is the actual macromolecule. Of course there are systems you can go to 10% of volume fraction. You have 100 milligrams per milliter but then it's unphysiological concentrations and it's not the real one you want in your sample. So I think it's very important for this time resolved studies to have these high flux facilities. If you do it in static mode and with medium flux you would measure such a sample for for half an hour. And since the typical typical time rates involved here are of the order of minutes, you need this very high flux. Okay, thank you very much. Frank for a very excellent presentation is very fascinating that you actually can can learn something for the structural evolution of the system and also you showed very nicely how to combine different techniques. So that's also a key for for what we are doing with with neutrons we need complementary technique and we should use neutrons to really pinpoint the chain this instructor that you can't get with any other techniques so so it was very nice. I mean just to say that there will be hands out hands out with the with the reference and everything and I guess you'll also give the maybe the email address of the speakers to the to the students I'm not sure so feel free to also write me an email if the students like to so.