 So I was asked to talk a little bit about fibre diffraction. In fact, I started up life in fibre diffraction for my PhD. I did fibre diffraction stuff on DNA, in fact, because my supervisor at the time was a guy called Watson Fuller, who did his PhD with Morris Bookings, who of course was one of the three people implicated, four people implicated in the DNA story at Kings and at Cambridge. So I started off doing fibre diffraction on DNA samples, looking at DNA polymorphism, because DNA is not just your standard double helix, which stays in one form. It changes between different forms, depending on the sequence in it and depending on the environment, the water content, the ionic strength. So I thought this was very interesting because obviously there was a temptation to, and I'm sure it's true in one way or another, temptation to think that these different forms of DNA had biological function. I'm sure that that is true, but it has never really been worked out. And so I'll try to give you a feeling for some of that, how we use X-rays to look at things changing, looking at DNA changing from one thing to another, and looking at using the neutrons to look at the water around it, which of course was one of the things that made it change, one of the things that drove transitions between different forms of DNA. But just to take you back to, oh gosh. Right, now I have no idea where, oh here we are. So fibre diffraction is all about filamentous molecules. So this is tobacco mosaic virus. It was one of the most famous cases of fibre diffraction, I guess. You know, that's an electron micrograph obviously, but there was a lot of interest in tobacco mosaic virus for its helical structure and for the amazing data that it gave. And amyloid fibres, many of you will know about amyloid fibres. There are hot topics in many ways, depositions that are deeply associated with particular pathologies. They are important in terms of increasing life spans and different types of pathologies associated with longer lifetimes. PF1 is a filamentous phage, a bacteriophage. These are just examples. This is the DNA protein complex. In fact, if the DNA, it's a picture I took ages ago, DNA complex to the RECA protein, which is involved in genetic recombination. There's DNA itself. You can see it there. You can see it in places where it's, you know, the double-stranded stuff has become a single-stranded and basically loops out. So filamentous molecules. Now, the key thing about filamentous molecules is that if you imagine, I don't know, a bowl of spaghetti. Let's just say you have a bowl of spaghetti. You've made a bowl of spaghetti. You put your hands into it and you pull the spaghetti. What you see is that all the spaghetti between your two hands will line up, right? Now, that's not crystallization, of course, but it's increased order. So you've lined up all the molecules parallel and you've introduced an order along the length of the alignment and which about that axis is randomly oriented, right? So that's what you could call cylindrical averaging, right? So you've gone from a completely disordered state to one where you have partial ordering in one direction, right? So that's what, in a nutshell, what fiber diffraction is. You've got to persuade your filamentous molecules, which don't crystallize and never will crystallize probably. You've got to persuade them to line up. If you want to move from a completely disordered diffraction data set to one where we have partial ordering and where you can get more precise information, perhaps, about the helical symmetry, but in the most favorable situation where you can get data that give you analyzable quality results and molecular modeling potential. Sometimes it's just that when you, you know, you can actually do parametric studies, you can watch transitions, as I was saying, you can watch things happening and you have all sorts of possibilities for doing particular things. And in fact, I just recently collected a data set with Marion across the road, across this opposite from my office, where we were looking at amyloid filaments and we were basically passing amyloid filaments from a tube through a capillary, which was in the beam, in the x-ray beam. And as they pass through the capillary, they line up and then you can get diffraction quality data. Those pictures are not actually in this presentation, but I wish I could try to find them. Anyway, so lots of interesting systems, not the level of crystallographic ordering that the crystallographers like to see, but nonetheless highly relevant to some of the most interesting systems you could imagine. And this, of course, is a famous picture and it's probably the most famous example of private diffraction and the consequences of this structure and determination have been inculcable as well as controversial in all sorts of different ways. But in that picture, you see Watson and Crick, you see their model, which was made of wire. It was a wire metal model. You can see along the outside, can you see my cursor? Can you see my cursor? Okay, great. So along the outside, you can see the sugar, those five-membered rings, that's the sugar group and then the successive sugar groups that phosphates in between them. And then in the middle, with torch stands, old style with torch stands, dimensioned and positioned, such as they represent the base pairs are going vertically upwards in that picture. And of course, we all know about that in different ways. If you actually, I think this is just, I don't know, this is probably a bit simplistic because I know you guys have, you've probably got lots of experience of diffraction, but I always like to show this because it was just, it's one of the things I found is a very interesting little teaching aid. But if you take this picture, actually came from the original Watson Crick paper, which in fact was, I believe, an appendix, a one-page appendix in Crick's thesis. So he had all of his other stuff, his human, whatever it was, oxygenated human globin and then had a one-page appendix at the end, which was the structure of DNA. So that's great. That's sort of quite amazing. But this is the picture from that nature paper, I think it was. And that's how it's represented. A double ribbon with a basis going between the two things. It was stereochemically, it was miles off. It was angstroms off, but it was right conceptually. And then the consequences for replication, storage, transcription and so on were all very much evident from this simple model. So, but if you think about that, you could think of DNA, you could just break it up into components perhaps, and you think of it as, how many of you have ever done a single slit experiment with a laser? Anybody? Anybody have one person there? Okay, so that's a few people there. So it's a very simple thing you can do. You can get a laser that you use to demonstrate, to use in a lecture theater, and you can take a single slit, you can put the laser through it, and you will see spots in a line across the screen. And that's the diffraction pattern of a single slit. And for the physicists amongst you, you will probably done this at school, done the experiment to measure the size of the slit and the variation of the pattern and this type of thing. And so what I'm just saying here is that look at this double helix, and just think of it as a set of slits in this direction. So that set of slits there. And think of another set of slits, which is like in the background of this picture, which would be perhaps like that. Just think of that in a simple, very simplistic way. I mean, obviously they're not evenly spaced. There's all sorts of different things going on there. And so you can sort of think of assembling the object like that. So if my slides are in one piece, then what you could do is say, well, let's consider the diffraction pattern of the red one on the second diagram from the left and think what would that diffraction pattern be like? So a single slit diffraction pattern, as I was just saying from a laser, this is what you'd get. You'd have your laser, you'd have a slit, you'd have a screen and that's what it looks like on the right. And you can adjust that pattern will change depending on what the slit dimension is. Also, on what the screen dimension is, is what the screen distance is and so on. So that's what it looks like, single slit diffraction pattern. And if you just consider this first part as a single slit or a set of slits rather, which is more like grating really, then you might expect something like that perpendicular to the slit, a set of spots like that. And if you consider the second one, you might, right? So that's just, it's a bait to teaching and it's just meant to illustrate that you don't have to get into the analytics of diffraction in order to exploit it or even for that matter to understand it in too much detail. Because in the end, this is the real DNA, as it were, or a space filling model. Those are the sort of slit concepts built into it and that's what the diffraction pattern looks like there. So that's the classic X-shaped diffraction pattern. I'll go into this in a little bit more detail but for those of you who haven't done do physics or haven't done math or whatever, don't worry about it. You can forget about it because I always think you should be able to understand and explain these things without a single equation. But I have got lots of equations later. But gloss over them if it's not your thing because what's more interesting behind all of this is sort of the implications for biology. So if you look at that, that's the X-shaped pattern that corresponds to, if you like, the sort of backbones and this simple model. And then, of course, perpendicular to that, you've got the bases going across. And that's another periodicity there. So that vertical periodicity in the case of the B form of DNA is these black lines here, the bases, base pairs, stacking one on top of the other. And so you've got another periodicity which turns up at the top of this diffraction pattern and that is, in the case of B DNA, the pitch of the helix is about 34 angstroms. There's 10 base pairs in one term. And the separation, of course, between base pairs is about 3.4 angstroms. And that's a very simple way of looking at this classical... We took this diffraction pattern off Darsbury. I think it's one of the Darsbury laboratory. One of the first experiments we did at Darsbury laboratory which no longer exists. But it's a very... It's iconic, this type of diffraction pattern. So what got me interested in the DNA was the fact that... I mean, this was all sort of known, that there was the excitement of the... When I started doing my PhD, the excitement of the fibre diffraction stuff and the DNA structure was still there, but it was sort of dying off and people were moving to single crystal studies of oligonucleotides, which were very interesting as well. But, of course, they weren't studies that related to the long polymeric molecule, which is what you actually see and what you get in real life. But nonetheless, they have their place in things. And so I don't know why I come back to this slide, but to just to summarise, the fibre diffraction is applied to... It's a natural one for molecules that assume helical conformations rather than the globular structures and their proteins. And while they won't crystallise, very often they can be persuaded to line up parallel to each other, which gives you the option of producing data, which cannot be analysed in the same way, but nevertheless it gives you data that may be the only way you get useable data and can give you important insight. Now, I mentioned spaghetti. And here's the molecular spaghetti I was talking about all in different ways. So if you imagine the first picture on the top here, that these two things on the left and on the right, those are glass rods. So basically you imagine a very thin glass rod that you've drawn in a flame and you take two pieces of it and you put the tip into a flame and then you make a little bead at the end of one little glass rod and make another same thing for the other. So you have this sort of sphere which is good for surface tension of the liquid you drop on and basically you set those up in a little jig and you put a drop of your DNA solution, DNA, onto it and then it dries down. So if you can imagine, I should have had a picture of it really, but anyway if you imagine a drop going between quite big at the beginning and then as it dries down you pull the glass rods away from each other and this is you and your hands pulling the spaghetti parallel to each other as it dries down as the concentration goes up. And then finally when it's dried and all the liquid has evaporated, you cut it in the middle and you've got two samples. So that's what you see there, that's DNA. This is a different way of lining up. This is TMV, tobacco mosaic virus. This is what was called a soul sample and it's where you get your concentrated virus sample and you literally flow it up and down in this capillary and as you flow it, it aligns. It's a sheer alignment. It needs to have a certain concentration. It needs all sorts of particular conditions and you can see it. Watch it line up. You can see by refringence and so on and you can characterize. That is outstanding and successful and used for many filamentous virus samples and different types of systems. And at the bottom, I show you the comparison top X-ray world, bottom neutron world samples. Big samples, small samples. So what have we done at the bottom? We've basically made several but many sometimes hundreds of individual DNA samples and assembled them to make a sample that's suitable for neutron work, big enough to get the diffraction button from a neutron source. So several things there, all based around molecular alignment and relevant to a whole range of different systems. Cellulose is another one. The most abundant polymer on earth currently. I can believe it as well. So cellulose is another good example we've done lots of work on cellulose. Now, so just to sort of put that in context, the application is fairly broad. It's not just the biological molecules I've mentioned. There's many biological examples. Collagen, there you go, another one. It's a hugely important molecule for connected tissue and so on. But also there's a lot of synthetic polymers and a huge range of interest there. We're surrounded by polymers in lots of ways. Polymers are, you know, modern world are plaguing us. It's all over the place. It's in the sea. It's inside animals everywhere. So for good reason or bad, polymers are very prevalent. And they're all filamentous and they all usually will line up in some way or another if the issue is to study them with diffraction. So cylindrical averaging. What do I mean? I think it's fairly obvious really. You've got your, if you have a crystalline, there's different types of fiber, right? You can have crystalline fibers, right? So where basically, and, you know, in DNA, you get crystalline forms of DNA. The X-shaped pattern I showed you earlier is what you'd call a semi-crystalline pattern. So you saw streaks making up that X-shaped pattern, which were actually close to continuous fully transformed. There's very little sharp spots there, but it was very well defined in terms of the layer lines you see in the pattern. But in some cases, you can get, I mean, I said, you know, we weren't talking about single crystals here. We're not talking about single crystals, but sometimes the crystalline blocks will form. They're microcrystalline blocks. They don't grow big into a normal single crystal, but you get small blocks, which are interconnected by molecules going between the crystal blocks. And they all are lined up for the reasons we've just talked about, but randomly oriented about the axis where they're lined up. So in the case of a crystalline fiber, the polymer molecules, they form the small crystallite, one axis aligned along the fiber axis. No preferred orientation normally about that axis. And so what you get in terms of diffraction is a bit like what you could imagine getting if you had a single crystal and you just continuously rotated it around one of its principal axes during an experimental data collection. All right. So it's cylindrically average, which means that some reflections, if you think about it, which in a single crystal pattern would not, would be symmetrically related, but which could be observed separately overlap when you rotate about that axis. So you'll have, you know, for example, I don't know, it depends on symmetry, but you might have a bar two, one, and the two, the two, one or something, they might, you might expect that when that you were taking them around the principal axis, they would overlap and form in the same space. So you've got to think about all of the different multiplicity and things in analyzing these patterns. So that's the crystalline fiber. And then there's a non-crystalline, you can get non-crystalline fibers as well, where lithium-ion, you don't think about crystalline blocks at all. You just think of rod-shaped molecules, if you like, that don't associate in any regular way, but which are randomly just randomly translated along beside each other. And that's where you get something more like the semi-crystalline pattern we saw for DNA. You don't have sharp spots, and you have something more closely relating to what I would call continuous free energy. Is that, does that make sense? You must stop me if I, if I don't make sense. It's, it's, it's been done. So just to give you a feeling there, what, what, when I say non-crystalline and crystalline, on the left is what I've shown you before. It's actually a different type of sample, but that's the semi-crystalline form. So you can see these spreeks, a lot of them, so vertically is along here. This is what I would call a layer line, layer planes, if you like. And so you have intensity sort of in streaks and not in sharp spots. When you can spread it to crystallize, it looks like this. So you get sharp spots, but you have this cylindrical averaging, which means that for every one spot, there may be a multiplicity of four, four, four, four, four, four, five, six, seven, eight. There may be a multiplicity of four or 12 or two or something depending on which part of the crystal lattice we're talking about. So that's the crystalline B form of DNA on the right. And there's the semi-crystalline, non-crystalline, if you like, form of DNA, BDNA on the left. So that's sort of cylindrical averaging. And, you know, you can, here's a graphical way of describing it. And so the circles on the left-hand diagram, so your reciprocal space of your reciprocal lattice is represented by that square picture there with all the various lattice points. And then the circles are designed to represent what the way in which the cylindrical averaging can be thought of. And there's the E-wall construction on the right. The central line in the middle of the diffraction pattern is traditionally called the equator. So if we go back to here, the equator is the horizontal line in this diagram or in this diagram. And then the vertical line up here, up this axis where you have all sorts of observations that you can relate to helical symmetry is called the meridian. So you've got the equator horizontally and the meridian vertically. And that's what's referred to in this diagram here, vertically and horizontally. So, and then, so I've mentioned here that when you rotate, you might get overlap, for example, of systematic reflections. Actually systematic reflections are usually okay, because you can divide by a multiplicity or something and account for it. Unless, of course, they're systematic reflections and they're not equal or a particular symmetry, in which case you have to refine against a compound observation. So, and then I've mentioned accidental overlap and accidental overlap is a sort of a strange term, but it means basically that they're not systematically related. I mean, like you take, for example, this crystal here, this representation of the crystal lattice or reciprocal lattice, and then you can see some reflections by the 001 and the 00 bar one would probably overlap and they might be of equal intensity, but you might get some that are very close to each other, which are not systematically related, but which start to overlap and then that's a bit of a nightmare to disentangle that, or again, you may have to work with a compound observation or close reflections. So, if you're going, if we just go back to, here you are, if you imagine, so they're one of those spots will have many components, but some of them are very close together and they have to be disentangled somehow. Anyway, so there we go. And, yes, so, and that's again the sort of, this is a PPTA, it's actually Kevlar, so this is the fraction part of the Kevlar, very, very sharp spots, some spots which will have, you know, systematic overlap, of course all of them will have systematic overlap and some will be very close, or as it were, accidentally overlapping. So, now this is where I've got, I have some equations, and I wouldn't, I don't want to label these too much because there isn't really enough time, but for those of you that've done the fraction theory, it's an attempt to summarize it in the case of helical diffraction. I'm not sure if the symbols are going to come out very well here because I haven't checked them, they were okay, but basically you can think of a helix being made up as a convolution of a single term with a point lattice having dimensions of the pitch length, so you're made to take one turn and then you repeat that several times vertically which is the pitch length repeated. And you can work out all of this theory, you always do it in cylindrical, you tend to do it in cylindrical polar, coordinates, and you basically, what you're trying to do is to work out the Fourier transform of your object, that's what you're always trying to do with diffraction. So Fourier transform of the object, I'll say it really doesn't matter whether you're into this theory or you're not. And so all of these calculations stress relate to the evaluation of that equation, and so we start off trying to work out this product here, r.s, in that equation and you do it in cylindrical coordinates for one turn of the helix and you've got it, that's the equation you come up with. I'm not going to labour it, I could certainly go through it if anybody's interested. And p is the pitch of the helix and we're just thinking here of an idealised single helix, it's a wire, we don't have any atoms in it, so this sort of concept is just a wire or we want the Fourier transform of that wire. And so you work it all out and you get this type of thing and that's all based on, we think about an infinite helix and it can be thought of, as I said, as a convolution of a single turn of the helix with a point that is having a spacing of p where p is the pitch. So you make up these concepts and then of course if you remember the convolution theorem you can relate the Fourier transform of each of those components as the product and what that tells you in the end is that the Fourier transform of the single turn gives you a set of planes having a spacing of one over the pitch, so everything is reciprocal space that's all about. So you work it all out and the point I want to get to is that it ends up being a set of vessel functions, this Fourier transform thing and then I just want to bring you back to the DNA because if you just, you go through all that theory and you work out the vessel functions and I don't know if you, how many of you will know about vessel functions but they're very simple, they're a little bit like damped sinusoidal functions and this is a J0, most of the vessel functions one, two, three, four, et cetera, et cetera, et cetera they start at zero and they have bumps like this and as you go, so this is a zero-thorner vessel function at the bottom you have first-order vessel functions and second-order vessel function third-order et cetera, et cetera now just look at the peaks there, right? They go further and further out as you go up higher order vessel functions and that's what I want you to compare to the DNA diffraction pattern because you'll notice that that's exactly what happens in the DNA diffraction pattern and that's why it becomes an X shape because every successive peak in the DNA pattern occurs further and further out as it radiates and in the case of the zero-thorner vessel function you have a massive peak at the origin which doesn't really concern us too much well it does actually but at the very origin it doesn't because we can't see the very origin because it's behind the beam stop anyway, so this is these are the vessel functions they the first maximum occurs at progressively larger radius or X in this diagram and they have gradually dimension magnitude as you go further and further out and further and further up and I hope I've got a picture here there we are so that's sort of what I was trying to say and of course you can relate that type of diffraction pattern to the DNA one so this is the zero-th layer line I called that the equator a minute ago this is the first layer line here second layer line, third layer line and the spacing between the layer lines is related to the pitch one over the pitch and so here you can see this very dark spot if we go back to our example or the vessel function it's in here, that's why it's so strong there's that vessel function there it's coming in about there so the general points are that the transform is restricted to layer lines given the symbol L and that it's cylindrically symmetric so you could rotate your sample and that sample would not change at all there are exceptions to that but that's the thing so then you say okay that's a continuous helix and we've roughly related it to the case of DNA but of course our helix is not really it's not really a discontinuous one it's got things in it it's got repeating units in it it's got a base pair nucleotide they have a base pair nucleotide which is here represented as these dots so let's just think of them as individual atoms because once we can do it for an atom we can add together all the contributions to multiple atoms and deal with it in a more simple way so along the axis then you have this repeating unit which has a distance of h in the case of DNA of course that repeating that distance would be 3.4 angstroms for a pitch of 10 of 34 angstrom pitch 10 base pairs per ton and so in the end you can think about this again if you forget this if you're not into the physics or the mass but you can think of this as a convolution of the Fourier transform of an infinite helix with the Fourier transform of a set of planes spaced by this distance h and so you can work through that type of theory and what that theory tells you is tossing over is a bit is that you would get as you went vertically up the axis in your diffraction space you would get a repeating x shape pattern every one over h every that repeating period of distance one over h that there's a thing called the helical selectural which tells you which vessel function contributes to which layer line and how they overlap and cause the diffraction expression here the vessel functions and the sum over all of the the different vessel function system now in practice you don't it depends on your symmetry in practice you don't see in the case of DNA you only see one of these extra x shape patterns that's the one over h that's the 3.4 reflection if you like here and you can see a shoulder coming down a bit and obviously in the centre you see the main one but they peg out as you go further out in resolution like all diffraction pegs out as you go further and further into higher resolution so there you are back to the DNA reference your x shape pattern your second x shape pattern which corresponds to the discontinuity and then you can see the shoulders coming off that there and of course you can imagine you've got vessel functions corresponding to each of these layer lines and then you have other vessel functions coming into the next x shape pattern if you like overlapping with the first set and you have to sum all that together in calculating the diffraction pattern for designing a target function for refinement so that's that's supposed to be a representation of a different you know the helical diffraction in a very very nut shell form having skipped over most of the equation stuff and these are other examples which show you the complexity of patterns when all of these all of these examples here there's no crystalline sampling whatsoever so it's all molecular transform and you can see here there's a the second set of vessel functions here starting to overlap with the first set of vessel functions and so on so you can see all these things they're not going to go into the details of indexing it but you can see how the system and you get quite complex things going on right now just to extend that a tiny bit further obviously we're not talking about a monomer that has one atom we're talking about a monomer that has several atoms in the case of DNA it's a nucleotide nucleotide based pair and you have several atoms so you have to sum over the contributions for all of those atoms and and in the case of x-rays we know we have the atomic scattering factor which is f in this equation and that has a certain form that you will probably know from other things depends on the number of electrons which atom it is in the periodic table and the form of this is identical for neutrons except for the fact that your atomic x-ray atomic scattering factor is replaced by your neutron scattering length and that's the only difference really but the calculation is otherwise exactly the same now we don't need to dwell on the calculations but that's just an introduction this is an old slide right you can tell that it's old because somehow old slides seem to be more yellow even though the technology is still digital but anyways an old slide and I was actually taking it was one I had a technician on the instrument and he had a piece to take me out in his plane and so that was a picture right to it years ago and anyway x-rays and neutrons that's what I was leading to with this equation here and that's the sort of the real life aspect of it the x-rays and neutrons and the river so that's the ILL I shouldn't need these pictures yesterday EMBL the synchron and so on is here's the scattering sort of summary and if you think of the scattering I've mentioned the atomic scattering factor the neutrons scattering lengths if you think of the x-ray case you don't need to worry about two lines but just take the second one as you go up in atomic weight your scattering amplitude goes up as well so at the very extreme you've got hydrogen practically invisible all the way up to to the higher atomic weight where everything goes everything scatters strongly and in the case of the neutrons as you go up the periodic table it looks like it's just random actually but at this end obviously we've got hydrogen and it's isotope that's important and then more or less of equal amplitude are most of the elements of biological systems and biomaterials you know, carbon, oxygen, nitrogen phosphorus and so on with a few exceptions like sulfur and things which are a bit different so that's supposed to your representation there and you've probably seen this slide many times you have x-ray and neutrons every time I copy this presentation into another file it seems to change the colours randomly but anyway so x-rays neutrons are scattering proportional to z in the one case and neutrons not proportional and then you've got these just random examples and then of course you've got in the case of neutrons you've got the isotopic replacement of H by D and that's a huge effect so as I said yesterday negative scattering length for hydrogen positive scattering length by deuterium and that shouldn't be thought of about negative scattering there's no such thing as negative scattering you what it relates to is actually phase of the scattered wave in the relationship to the instant phase but nonetheless in the density maps you will the negative peaks will give you negative peaks in the map and that can cause difficulties based on all by deuterium and things much easier but you can see how that compares hydrogen then becomes similar scattering power to carbon oxygen they're all in the same ballpark so that's the x-ray now I'm not going to go into the incoherent scattering cross sections because I think we talked about incoherent and I suspect Frank and others will have mentioned this but the only thing to take away from that is this is supposed to represent the incoherent scattering hydrogen versus deuterium so you can see the massive difference here hydrogen deuterium and that's very significant and I tried to explain that yesterday in terms of the background that you saw and that restricted the the measurement of the Bragg spots in your crystallographic experiments and the other thing to remember is that if you look at this sort of x-ray scattering and I can't remember what that is carbon I suppose the x-ray scattering factor as a function of angle you know the fraction pattern as you go out in the scattering angle it falls off the x-ray scattering factor falls off as a function of scattering angle and the reason for that of course is that your electron cloud is of the same order of magnitude as the wavelength of the radiation you're using to probe it so let's just say your wavelength is one ounce strong or one and a half ounce strong and your atoms are in that ballpark so you have an object and this comes back to the Fourier transforms again if you have the larger your object the more quickly the electron will fall off but in the case of neutrons the nucleus is essentially minuscule compared to the wavelength of the neutron beam which would be about the same as the x-ray beam wavelength and so it doesn't fall off at all so it's a straight line so in principle there's your neutron scattering length and it just goes on of course it does fall off and what that means is although your neutrons are low flux compared to the x-rays massively the x-rays are massively more high flux than the neutrons the scattering length doesn't really fall off over the deflection time you observe and there may be some pictures that I'm not sure of so these are some summary patterns summary facts about x-rays and neutrons you know typically you know you get various things there you can get high resolution with both but often your definitive molecular structures come from x-rays small samples, time resolved dating you've got so much flux that you can watch things changing and there are sample damage issues of course, radiation damage and then neutrons low resolution in solution contrast variation as you've seen high resolution for graphic experiments where you see hydrogen and then fiber diffraction for example water, hydrogen essence, filamentous molecules so that's in terms of dynamics which is something and no sample damage now back to DNA the reference molecule there you see just to bring the neutrons in the neutron fiber diffraction putting it alongside the x-rays so on the left you've got the x-ray diffraction, fiber diffraction pattern of the B form of DNA and then on the right I can't begin to describe how we actually did this experiment because it was so long and the sample was so hard everything was hard about it but anyway, on the right you see the same type of sample obviously much bigger because it was neutrons, the bigger sample hydrated in H2O and then what's all that's happened here so when you do DNA you may remember from history books or papers and stuff that you have to keep the sample shielded because it's a water and then of course what you have to do in studying it was to bring the humidity to a particular value that optimised the quality of the diffraction pattern and actually that was the way you induce conformational change as well so when you did experiments you were always recording data in a humidity chamber and you were always recording data in the presence of H2O and the whole idea of the initial experiments we did on DNA neutrons was we were just doing the simple thing of replacing the H2O in the environment by D2O and so you can see there what happened when we did that and I say this was a big task if you look at these pictures you'll see strips in there and those strips reflect the size of the detector in comparison to the situation of X-rays where we was getting all in one shot on one detector we had to record individual strips for the entire diffraction pattern moving the detector around and so on and then assemble the whole thing into a contiguous image that gave you these patterns and on the left you see the neutron pattern with the DNA hydrogen H2O and on the right you see the neutron pattern recorded in D2O and the difference is huge and that is solely attributable as you can see all over the traction pattern everything changes and that is solely attributable to the water that's located around the DNA in a structured way structured water around DNA and that was what we were after at the time so now I come to this I don't know whether my movies maybe they are maybe not anyway I wish I could show you that anyway I come back so neutron X-rays you see all the things there and I'm just going to sort of summarise some of the things you see when you try to change DNA forms so on the left hand side in the middle we've got the B form of DNA and this was for a very particular repetitive sequence DNA it wasn't random sequence DNA it wasn't carbon dimers DNA it wasn't salmon sperm DNA it was a very particular alternating AT sequences which actually are quite important in biological in DNA function and structure but anyway the aspect of repetitive sequence of DNA that's striking is that it gives you a completely different form of DNA called DDNA which you don't see in single crystals but you can obviously see it's highly crystalline from this pattern instead of having 10 base pairs per ton it actually has 8 base pairs per ton and anyway so you can get this thing to change between the two forms and by just altering the humidity it was supposed to be a movie cursed but it shows the thing going between one and the other and that was totally using the high flux of the synchrotron to be able to follow this thing in time-developed mode so I mean if we ever have chance to talk again about this I can certainly dig out of those movies so you can see they're very spectacular so that's one example of transitions exploiting x-ray exploiting flux being able to do time-resolved work and that's of course a massive I mean actually too I'm probably going on about this but the other thing was fibrous molecule that actually drove synchrotron radiation into full exploitation was muscle because all of the people in the early days they were all after x-ray flux in the early days there was the crystallographers trying to solve all these amazing proteins Dorothy Hodgkin and all these people but then alongside those guys there was a huge interest in trying to understand the contractile cycle of muscle and that's where they wanted muscle they wanted flux that's how rotating anodes came onto the scene after fixed anode systems and that's how the first synchrotron radiation sources were propelled they weren't actually propelled I don't think by what you might assume now the sort of protein crystal they were propelled by muscle and then of course once the muscle then the muscle was hard you got to get to millisecond resolution to look at the muscle contractile cycle but then obviously the crystal they took off as well so history is interesting of all of that but it probably wouldn't have happened in the same way without muscle anyway there's another one here that I mentioned that's gone wrong on the slide there's one left-handed form all the DNA forms part one is right-handed but the Zed DNA is left-handed that was actually first discovered in a single crystal study and you can get this is the intriguing thing you can get right-handed BDNA turning into left-handed Zed DNA with 12 base pairs so completely different steric chemistry completely different geometry and these pictures on the right which seem to have been marked up a bit are our sequences from the diffraction sequence recorded as it goes from one form to the other in this particular case which involved methylated DNA 5-methyl cytosine it's the one way once you've gone into the Zed form you can't get out of it again so it's trapped probably by the methyl group so anyway so I just make these points about the time resolved aspect of it and coming back to the neutrons as well we sort of used these differences between the D2O based DNA work and the H2O DNA data to do Fourier analysis of where the water was going and we get pictures like this and now this is a different sort of thing it does follow, the analysis follows fairly crystallographic patterns but what you see here are very clearly structured water patterns in two different forms of DNA so this is the D form of DNA you've got very highly structured water minor group of the helix and then the A form it's the opposite, it's the major group and you've got these regular water bridges formed as you go down but linking the phosphate oxidants so water driving the transitions X-rays watching the transitions and neutrons telling you what the water is doing of the bottom line there now I realise I'm going on in time but I wanted to at least let you know let you see yesterday I showed you a diffraction pattern where I simulated the consequences of hydrogen incoherent scattering and the background and the visibility of the spots and so on and this is one of the things that you can also see in the DNA, this is a neutron experiment where you start off in 100% H2O so again look at that background you can see some spots here I don't know what you can see on your screen but you see some dark spots there which are actually the coherent diffraction from the DNA and then this background stuff is the incoherent, the dark stuff and what we did was we simply changed the supply of vapor around the sample from H2O to D2O and we watched it change and as you did that you see the background change and so your visibility improves massively I'll just go through it again there you go D2O replacement H2O background improving signals and noise of the days you were after improving as well so yeah so and then these are sort of comparisons now I made these points this was all DNA that had not been converted so although we were talking about replacing the solvent around it by H2O by D2O there's still all of the hydrogens left covalently attached to the DNA itself to the sugar groups for example some on the bases because this was not DNA grown in deuterated media it was just DNA in which the solvent had been replaced to vapor exchange in the environment of the sample but if you look at that DNA and I hope this picture works these are all of the ones so this is a DNA for example without hydrogen and then here are the hydrogen atoms that remain to be replaced and which you can replace if you then grow your DNA in your grow cells in for example you can grow them in deuterated media and then you can extract the DNA from that and then you have deuterated DNA so that's a summary of the coherence and incoherent diffraction and I think I'm probably going to stop there before I get on to contrast variation because I think Frank is probably covered contrast variation and I think I'm already at 55 minutes is that am I right Tommy are we yeah it's fine so stop there and take some questions I think so is there any questions for Trevor Jenny has raised her hand I see Hi I was just wondering with the changing in the changing from D2O to H2O or the other direction obviously there's always kind of the concern that the of your sample actually changes when you go between them how much would you say that's actually an issue and does it vary what you're looking at so you're talking about basically an isotope effect yeah so absolutely you're absolutely right and you should always be thinking about this whether you're doing these insolutions or whether you do whatever it is you should always be and I would say in particular you would need to be a little bit careful about lipids in fact usually lipids and so on and so yes so but what we do anyway let's just say we're looking at a we have a deuterized structure, we have a hydrogenated structure and we want to be sure that they're okay so what we always do is just we do the x-ray structures of both and that would be this case whether you did it with DNA your deuterized stuff you want to compare now what we find is that mostly the structures are closely comparable and not always, every now and then there is a problem but what should so just to come back to DNA which has been the reference to one of this which is sort of good if you follow a conformational transition if you take random sequence DNA it goes to a sequence it goes from C to A to B that's the transition and that is highly the humanities at which that occurs is highly dependent on salt content and what we found was that in the case of perduated DNA everything replaced that the deuterized stuff occurred at different salt concentrations so the transition shifted in terms of ionic strength and that probably comes back to deuterium and electronegativity and all sorts of things that I really don't know is what I understood but it's but you're absolutely right if you're in this business you have to constantly do these checks and mostly for us what we do is we cross check with X-rays okay there is another comment from Susama let us yes I have a question so for the DNA studies is it a synthetic DNA or you extracted it from bacteria or something? both so well there was a whole lot of different things so the one I just referred to with Jennifer that I was referring to what we used to call random sequence DNA which is natural DNA that might be extracted from carthymus or salmon sperm or clostridium perfingions or something but when I referred to the D form DNA that was a synthetic DNA alternating ATC somewhere I have a diagram that summarizes DNA polymorphism which is incredible so the D form it's only observed for alternating AT and the one where I mentioned the Z form or the Z form as the Americans will say is actually alternating GC and the interesting thing there is that it was methylated so you methylate DNA if you have size to see methylation which is associated with transcription effects the behavior shifts but you still have this left handed form so in the data I showed you there was a combination of synthetic and natural sequence okay and how long are those DNAs like how many base pairs? oh tens of thousands of base pairs so you multiply by 3.4 it's huge okay into the microbes so maybe I can ask another one did you try to deuterate DNA but like short sequence DNA like 100 base pairs sorry just say it again if you managed to deuterate DNA but like short DNA like for instance 100 base pairs or even less you're asking me could you or if someone did it because I tried to do this and I failed so I had a complex protein DNA and my DNA now it's 60 base pairs oh I see so short oligos you were talking about yeah pretty much so obviously you can but what you have got to get hold of is if you want to deuterate it then what you've got to get hold of is you've got to find somebody who's got a an oligulite synthesizer and get hold of the deuterated phosphoramidites that you have to feed into that synthesizer in order to get your deuterate stuff so it's a little bit non-trivial and you need and we've got a little bit of a project going on at the moment with the sorry content biology centre where there's a student who's trying to do exactly this to produce the synthetically produce the deuterated phosphoramidites for DNA crystal but yes it's a pain it's an absolute pain it's not something I mean we were doing it if you like it wasn't easy but what we were doing was deuterating stuff from coli cells then phenol extraction and purification of polymeric DNA not the same as what you're talking about where you want presumably a rather specific sequence that's 60 base pairs long or something so yeah it's it's something that needs to be developed I would say yeah because we are thinking with Antonida from Anstor just to do this in E. coli so we had a synthetic plasmid and then we put like 20 repeats of the DNA sequence and then we just try to like gigaprep this yes you say you started and you failed or this is another idea you said did you say that you had tried that and failed so we actually we didn't manage because it was during corona time so it failed everything failed in corona time yes it's a bit of a problem and it would be great it's always something I felt that we should do is to develop the problem really is in the end that you know if you run a DNA synthesizer you need somebody dedicated more or less to run it we do peptide synthesis like run a DNA and then you've got to get the protected nucleotide things and yeah it's just a bit of a bubble really and then you've got to somehow keep it running and so it's difficult yeah okay thank you maybe one day okay is there any other questions for trevor and question about the alignment I mentioned it's very important to have perfect alignment fibers well you get you get I'm trying to think now I have so many slides no I think alignment is an issue I mean obviously you never get perfect alignment any more than you get a perfect crystal but and sometimes the alignment is dreadful for some systems and sometimes it's very difficult to optimize and sometimes it's unusable so yes it's a absolutely principal concern but you'll see the DNA ones that those are pretty well aligned and there's lots of cases where alignment is very good and and so on what's actually happened what's happened what we've done more recently and I haven't I've got data in that PowerPoint somewhere on it but is where we've been using the fell at Stanford to shoot Fibrella particles through a jet they line up in the jets through shear alignment and then you do serial fiber deflection on them but in principle one pulse is hitting one particle and then you're getting each of those crystallites in the crystalline diffraction pattern being separated and then you can perfectly or if you like the word perfect because you can take each diffraction pattern and oriented computationally the data and then assemble the whole thing if you want to see what it looks like and it looks beautiful actually so that's decomposing the fiber into its constituent contributions so I for an hour and a half I would have got onto that thank you very much I guess Travi you have this special spinning device for the DNA stuff or well actually we do have it it hasn't been used for a while that's Adam Andrew Wiles was the guy who was doing that and I think it still exists but he has moved off that and this came from Alan Ruprecht in Stockholm I can't remember and he pioneered all of that he used it for DNA but he also used it for polysaccharides and so that was a pretty heroic thing he did but it's sort of when he passed it on to us in his retirement and Andrew has done a fair bit of it with Michel Perra and other people and so yes we have it available but nobody actually has a project for it at the moment yeah that I remember as quite an inst great job orient thing maybe I can ask a last question so I was just thinking about this coherent and incoherent scattering you showed like so do you consider like the incoherent scattering at all or do you just can you get any other information from the incoherent scattering or is it just only a background yeah that's a good question and the answer is yes in principle if you have the right instrument you can use incoherent scattering to get mean square displacement of the atoms causing it but I don't know if you have any dynamics guys will tell you that I don't know if you have any dynamic structures I'm not really a dynamic person but in principle what happens is your hydrogen incoherent scattering as a function of the scattering angle falls off and it will fall off more rapidly if your mean square displacement is low then in principle you can extract the problem with doing it in a diffraction experiment like that like hot neutron and fast neutrons and it's a complex so you need to have a more dedicated instrument that can measure the incoherent scattering so it should not the word noise is sometimes used to describe incoherent scattering and that's not right and the dynamics people will refer to it if you call it noise because for them it's their signal so there's information there and it can be exploited and we shouldn't forget that either the other thing is that in a wonderful utopian world you can use a magnetic field in low temp to separate the spin states of hydrogen and completely eliminate hydrogen but that's not easy yeah and this was like maybe follow up question like then there is a difference between doing this in x-ray and neutrons because we said that neutrons have like a stable amplitude yes with angle yes exactly exactly