Dorothy  Crowfoot Hodgkin (1970) - Structure of Insulin

Professor Kunitz, ladies and gentlemen. It is a very great pleasure to be here today and I must thank Professor Kunitz for all of his kind words and particularly for his very useful introduction to the subject of my particular talk. Insulin, as no doubt you all know, is a hormone that is responsible for part of the control of the metabolism of sugars. And in its absence the disease diabetes intervenes. It was first isolated in 1921 by a group in Toronto, Macleod, Banting and Best, of whom Best at the time was a graduate student aged 22. And they were able to isolate it following first of all the much earlier evidence that the active hormone occurred in the pancreas and that it was a protein. And so they were able to take effective measures against the protein being digested by the enzymes that occurred in the pancreas. The next stage in the story, as far as this story of mine is concerned, was the crystallisation of insulin in 1926 by Abel. Abel actually was professor at John’s Hopkins, aged 67 and near retirement when Professor Noyes produced a small sum of $10,000 and invited him with this to Pasadena for a year to try to crystallise insulin. And the $10,000 allowed him to collect a small group of graduate students to help him. And in the course of the year they obtained the first crystals of insulin and a picture of these is on the first slide. This first picture is taken from Abel’s notebook and you see he looked up mineralogical texts and was able to describe the crystals as trigonal from Dana rhombohedral class. Later he was able to obtain them looking somewhat more like the form that we usually see, little roms of very characteristic flat shape. There was some difficulty for two or three years over the crystallisation of insulin. Squib insulin crystallised and Lilly insulin didn’t. And this was traced to the occurrence in Squib insulin of a small amount of zinc, which is necessary for the appearance of this particular modification of insulin. The existence of zinc was shown by Scott in Toronto, it does also occur in the pancreas. Scott found that the same kinds of crystals would appear with certain other related metals. Now, once the zinc was found to occur and be necessary in 1934, of course all the different firms making insulin could obtain insulin crystals. And a small sample of insulin crystals was given to Sir Robert Robinson in Oxford by Boots when they first made it. And because he had no real intention of working on this subject himself, he gave them to me. I had then just come over from Cambridge where I had been working with Bernal and had been involved with him in taking x-ray photographs of the crystalline enzyme pepsin. And Robinson thought I might try to do the same, take photographs of insulin. So I studied this back history and tried the different methods that they had used to grow insulin crystals large enough for x-ray work. And I must say that one of the most exciting nights of my life was when I saw the first spots on a photograph produced by passing x-rays through a crystal of insulin. Now, the next picture shows you an x-ray photograph of an insulin crystal, very, very much better than the one that I first saw. But you can see the reflections here have a definite hexagonal pattern of intensities and a definite distance apart. And from the distance apart of this photograph, combined with others that cover the whole diffraction picture of insulin, one can calculate at first just the size of the repeating unit. The size varies a little, according to whether the crystals are wet or dry. And I will put up here the later measured value for the side of the rom of 49 Å and the angle 114½ degrees. The first calculation that one can make ... (laughter) The first calculation that one can make is what is the weight of protein in this particular volume defined by the unit rom, and allowing for some 30% of water of crystallisation, the weight is just round about 36,000. This seemed dramatically close to the weight of the protein molecule in solution found by the first experiments of Svedberg in the ultracentrifuge. He gave a weight of 35,000 as the molecular weight of insulin. I was a little bit concerned, I discussed the problem with Harington, who worked on insulin, that in fact these crystals are trigonal and that this weight should therefore be divided into three equal parts, And this brought an immediate letter from Freudenberg in Heidelberg, saying Svedberg doesn’t really measure a molecular weight, he measures a particle mass. And the amount of chemical change that inactivates insulin is so small as to suggest the molecular weight is much smaller than 36,000, in fact he gave an estimate of perhaps from 9,000 to 18,000, which of course was nearer to the possible 12,000. But, as the years went on, it became clear that the molecule of insulin was smaller still. There was evidence that the particle of 36,000 would break down in solution and gradually the evidence accumulated that the weight might even be not 12,000 but 6,000. Though that this was so was not really clear until, on the next slide, Fred Sanger found in detail the structure of insulin and I’m sorry this is a little bit small to read from a distance, as I realised when I looked at it a moment ago. But essentially Sanger showed that the insulin molecule consisted of two chains and the amino acids of which luckily you were shown larger pictures today, are here given their abbreviated names leucine, valine, tyrosine, alanine. And a definite order along these two chains, one of 21 residues, one of 30 residues, joined by disulphide bridges at particular points. And of course, as soon as this structure was produced, this chemical sequence, it became possible to synthesise insulin. And three different groups set out to synthesise insulin and succeeded, Zahn in Aachen, Katsoyannis in Pittsburgh and a group of Chinese scientists in Shanghai and Peking. And the next slide shows a little bit of what is involved in this process. This is taken from one of their papers, they all synthesised separate peptides, different choices in the different laboratories. And when they had synthesised the A chain and the B chain and the cysteine residues at the right points, they reduced to the SH form and then left them in solution, hopefully together, in the presence of air and some insulin formed. Not a good organic chemists synthesis, just hoping that nature would work and insulin would form out of these separate chains. But it worked to a small degree, the first products got by this reaction had only a few percent activity but out of them, by suitable manipulation as on the next slide, crystals of insulin could be obtained. Again, not perhaps such very good looking crystals, but you can see this very characteristic shape. These are taken from the Chinese paper. Now, so you might say we know the structure of insulin, but of course it’s a problem when one thinks of it, these two separated chains, how are they really disposed in three dimensions. And throughout this long period from the isolation of insulin and our first work, we really hoped to find the actual 3-dimensional arrangement of the insulin molecule in space. We grew very much better crystals than the first ones, these are actually very beautiful crystals taken from a paper by Schlichtkrull in Copenhagen, who had developed crystallisation techniques to a fine art because he used actual crystalline insulin, very finely crystalline insulin as the basis of a method for giving slow acting insulin to diabetic patients, the novo insulins. He also found that in these particular crystals there were in general two atoms of zinc per 36,000 molecular weight. How to find in detail the arrangement of the atoms in space given these crystals was in principle of course known when I started out in 1934. The next slide gives a letter, a little extract from a letter and I think Professor Ruzicka may recognise the writing, although the signature isn’t there. It’s J.D. Bernal. And he was very excited because of the x-ray photographs of insulin and he immediately suggested that one should change the zinc for other, heavier atoms. And the implication of this was that we should be able to calculate the actual electron density in the crystal if we could observe changes in the intensity produced by these substitutions and have an isomorphous series. Now, actually these are the substitutions that Scott had made in Toronto but the cadmium is rather little much heavier than zinc and the actual differences that I could see at that time were far too small to work on. Also the method was untried for very much simpler compounds and it seemed desirable to have some simpler subject from which to try out the calculation of the electron density by isomorphous replacement before we tackled insulin. The next slide just gives you, I think, the background relations involved the actual form of the calculation of the electron density in a crystal, at a particular point, FHKL being derived from our measurements. Both FHKL and the phase angle appropriate alpha depend upon the positions of the atoms in the crystal. And if one can vary amongst these atoms, that each has its appropriate scattering number, and introduce heavier atoms, one can formally solve for the phase angle and this of course was what we hoped to set out to do. And the first substance, I tried this on in a very simplified form because the certain parts of the distribution was centrosymmetrical which simplifies the phase relations was penicillin. And I think the next slide just shows you the look of the electron density in penicillin, set up in a way that we used here for the first time but has become the way that is used in protein crystallography today. We calculate the electron density, a series of figures at points over 3-dimensional space from the measured intensities of the x-ray diffraction effects. We join up, we plotted out on sheets, first of all in two dimensions, join contours of equal electron density to get representation of the atomic positions and so form a picture of actual individual atoms on sheets of transparent Perspex or glass or other material. And here of course you see separate atoms resolved and on the next slide the formula that you could write as a result of this vision of the individual atomic positions in space. And we were lucky that penicillin is at least a little bit connected, derived from a peptide kind of skeleton. In the case of insulin as with other proteins, although the diffraction pattern that you see may look marvellous to your eyes, it doesn’t really extend sufficiently far in space to resolve individual atoms. The next picture shows you the consequences of this, illustrated by a very simple molecule. Could I have the next one, please. If the space, in penicillin the actual pattern extended to about .8 of an Å resolution. This shows diketopiperazine seen at gradually reducing degrees of resolution. This being the stage at which most proteins reach. And at least a little bit better than the case of the insulin maps we were to calculate. You can see that some atoms almost appear individually, but in general there is a lack of resolution and one has to infer the positions of the atoms from the very characteristic shapes of the electron density peaks that one can calculate in a protein electron density map. Now, our real trouble with insulin turned out to be twofold. I must say I was trying to introduce heavy atoms into insulin crystals parallel with John Kendrew and Max Perutz at Cambridge in haemoglobin and myoglobin. And we had no single chemical point at which we could attach a heavy atom, as in the case of haemoglobin, but we thought, perhaps therefore we should follow the line found by Kendrew and try to get just heavy atoms to pass into the crystals by other means. And actually it was Carlyle working with Bernal who first produced the idea of floating them into the crystal by just soaking them into solution. So we tried a very great many experiments and we found it very difficult to find where the heavy atoms were. And here I have to start to go back a stage with my formulae. The first thing one can calculate is not of course the electron density, one can only begin to calculate the Patterson, taking the F2 values and obtaining a derived function of the electron density. Well, the next slide, I think, should show you a Patterson map for insulin, this is just a bit of the 3-dimensional Patterson function. And actually it’s quite interesting, we started by thinking we could know nothing at all from it, but you can see something from it. This is just part of the function and in this pattern you do see in fact very roughly plains of symmetry which don’t extend the whole way through the crystal. But a very complicated pattern. The implication of those plains of symmetry is shown in the next slide. It is that in insulin, in the unit cell we have in this 36,000 mass six molecules. These six molecules, of course three are arranged around the 3-fold axis, but the other triplet is arranged so that there’s a twofold axis relation approximately between them. And so over here the Patterson function, derived from this, shows plains of symmetry. The Patterson function is got by imagining you stand at each atom and put each atom in turn at the origin and plot the whole function round it, so it’s derived from it but much more complicated. At this point two things became obvious. First was that if there were only two zinc atoms in the crystal, as Schlichtkrull had found, they must lie on the 3-fold axis and that was one great help. And secondly that we would expect that other heavy atoms introduced into the crystal should have this characteristic pattern. And so if we did a difference map, taking, measuring the intensities of the crystal and the heavy atom, we should see this sort of pattern in the difference map. Now, the first point was quite alright, two zinc atoms on the 3-fold axis. The second turned out to be a small hair, they seldom if ever showed anything like the correct symmetry for reasons that are now obvious. The next slide I think just shows one of the difference Patterson’s of the heavy atoms, a very complicated looking one but the little peaks near the origin do imply that the heavy atom in this crystal has gone in near the 3-fold axis. Now, the heavy atom in this particular case was a very interesting one. It was got by taking zinc insulin crystals, leaving them over night in EDTA solution, this removed the zinc from the crystals and left us with crystals standing looking rather sorry for themselves but still giving x-ray diffraction effects. And if we then left them in lead over night, the lead went into the positions occupied by the zinc and also into positions represented by this map and into some others on the outside of the molecule. And I will now cut a very long story short of how the different heavy atom positions were found and take you essentially to the answer. So on the next slide I have first of all the positions found for four different, five different heavy atom containing crystals, plotted along the 3-fold axis over a unit of volume which covers a whole insulin hexamer. And you can see a lot of them come around the 3-fold axis. The pattern doesn’t have twofold symmetry, there seem to be two general sorts of positions, one near the 3-fold axis and one out on the edge of the molecule as we later found it. And our difficulty was the sorting out of this very complicated pattern of heavy atoms before we could calculate the phase relations and the 3-dimensional structure of insulin. But now we pass to the actual electron density map. So the actual calculation came through the middle of July last year and we plotted it out and drew it up. And I won’t show you the original map because we have drawn it all much better since then, but the next slides are taken from a little model, very like the penicillin model but representing the electron density as we calculated it over the insulin hexamer. And because it’s a large molecule and this hexamer, this is 30 Å deep and 46 Å across, the actual little blocks are broken up into 5 or 6. And I will just run the slides through so that you can imagine yourself looking at the molecule from top to bottom and thinking what you could see. The next, ... this is the top of it, looking a little bit empty, rather complicated set of chains, these are half a dozen sheets projected together. Now, on the next, could we pass on to the next one. Now you see we’re thickening up tremendously in one particular region, a lot of peaks coming together. And in this part there’s the beginning of the zinc atom and these three peaks are three peaks that are above it. And the next slide, it’s becoming a bit more understandable. Here’s the zinc here and now these three peaks are three peaks that are underneath it. And they seem to run into a very definite chain structure here with peaks coming off it at certain intervals. And at this point we began to be looking at Sanger’s sequence and thinking what we might be seeing. Because we knew that there was chemical evidence that the histidine groups of insulin interacted with the zinc. And so we looked at what would be near the two histidine residues and in one case, histidine 10, if we take this as the histidine and come on, on one side of it it goes to a serine coming back towards the zinc and on the other side it goes out to a leucine and here is a peak with a kind of umbrella end that could be a leucine. And from this point we started tracing through the chain. Now I’ll just show you the next two, this is passing below that particular point and here we come to a highly empty region in the middle of the crystal with chains coming in towards the central cavity. And this is the central cavity into which so many of the heavy atoms went in a somewhat random way in our substitution. And the next one passing down below this central point, we begin to see another similar chain, the histidine on the other side coming in, the leucine on the other side, the serine on the other side. And they don’t look absolutely the same but they’re sensibly the same sort of object. And then, down below that, the zinc with the other three little groups above it and the beginnings of the bottom of the molecule, the other side. So now you’ve passed through it all and in about a week we had traced very rapidly where we thought everything was in the insulin molecule. Then we’ve taken a year going through everything, not quite a year actually, it isn’t, it’s only about eight months, isn’t it, going through everything much more carefully. Comparing the electron density map directly with a much more accurately built model, a model 2 cm to the Å, which we could project directly into a map of the electron density at the same scale by a mirror device produced in Oxford by Professor Fred Richards from Yale when he was visiting there for which we call in Oxford Fred’s Folly, which doesn’t mean a folly in a foolish sense but because a folly is a special kind of little home or house, a delight you see for all of us who work with it. So on the next slide, I think I have a little bit showing you how closely you can in fact fit the appearance of the residues to the shape of the peaks, as they have come out in this 3-dimensional electron density map. And at the present moment we have passed the whole way over the molecule and recorded the positions in three dimensions to a first approximation for every atom in the molecule. So the next few slides, if I might have them, just show you the look of each chain in turn, all the time projected in the same direction along the 3-fold axis. And now this is one of the B chains of Sanger’s molecule and it starts up at that end, B1, which is a phenylalanine group and it runs in an extended form as far as this V10 histidine which comes into the zinc. And then it turns through three turns of a very good alpha helix. And then it runs right down and right up in another extended chain. It’s better that afterwards you look at the model because then you can see what's happening. You come along the back, you go into this alpha helix, straight down underneath and then you come up in a long chain right up here to the top again. And now the A chain, on the next slide, just sort of nestles in to the B chain, it just forms a little closed loop. You see starting here and going down there, just lying on the face of the B chain. And the disulphide links that link the two chains together, link them, one at this end to the alpha helix below and the other one at the other end to the other end of the alpha helix. So that the alpha helix, helical part of the B chain forms a kind of rigid skeleton on which the A chain lies. And now, over the other side, I’ll show you the other two molecules. This is the second molecule in the asymmetric unit. Again the B chain starting out a long stretch of extended chain, the alpha helix coming down here and then we’re seeing this long end of the B chain coming up on end. And on the next slide, the second A chain, again forming this small, very compact loop within the, over the surface of the B chain. And now you put these two together and in the next slide you have the same projection of the insulin dimer with the two molecules together. And I think you can begin to see that whereas this line is an approximate twofold axis, it isn’t by any means an exact twofold axis, that there are slight differences in the relative positions of the groups on either side of it. Now, the actual shape of this dimer, and I’m sorry that I haven’t got it here in bodily presence before you, is a rather elongated oval. The next slide shows you a view along the side of the dimer as seen in this more accurate model that we have in Oxford which the boys won’t let move away from Oxford, even to be shown at the Royal Society at the present, because they regard it as such a delicate object that has to be preserved in the exact form in which they have built it. But anyway here is one molecule and the second molecule packed together with the two zinc atoms along the 3-fold axis, each of which has directed at it a histidine residue. This is viewed from the direction of the zinc atoms and the next slide shows you the view from the outside of the molecule. Now, if you could, perhaps we might have a little dimming and the rest of it at this stage. This shows you that these two molecules are fitted together in a very intricate way. The main groups that pack together are non-polar residues but there is a very long region here where hydrogen bonds form between the extended ends of the B chain. This is the extended end from one molecule going up in this direction and this is the extended end of the other one coming down in this direction. And the hydrogen bonds form across here, you can see them in red so that this is part of an antiparallel pleated sheet. And it’s between the two insulin molecules. Now, the next slide shows this just drawn out so that you can see the way that it goes with the hydrogen bonds forming on either side of the twofold axis in this generally antiparallel pleated sheet form. But now, if we look onto the twofold axis, that is to say the way we’ve been looking most of the time, we can see that while these groups are quite nicely related, the individual residues are not. Could we have the next slide. This is looking onto it and now the residues that project are ones like here are the phenylalanines and really these phenylalanines to be related by the twofold axis, one should turn away in one direction and the other one the other direction. But in fact the second one turns across it in order to pack side by side nicely as phenyl groups like to do, disregarding this twofold symmetry. And up here again the valine groups don’t seem to have the same orientation and again it looks as if they just turn to fit nicely into one another, there’s a non-polar interaction at that point. And at the end we have the B13 glutamate ions going in to the centre and they are the ones which attract these heavy atoms into the central pool of the insulin molecule. Now, the next slide I think shows you, just to give you an impression, to go on from the dimer to the hexamer. This is a projection of all of the atoms in the unit cell of insulin as so far placed by us, that is to say the atoms belonging to the molecule. And you can see that these six insulin molecules have formed a very good spheroidal object. And these spheroidal objects pack together, in the next slide, in a way that just is clearly quite good close packing of, it’s actually cubic body centred close packing, but you can see the way these spherical objects, the insulin hexamers fit together in the crystal structure. So now we should think in more detail about the complications of this molecule and what it is doing in nature. And this is still a very great mystery. We have this very complicated arrangement of chains within the molecule which yet ends up you see producing a very remarkable smoothly spheroidal object. And this smoothly spheroidal object, it seems, does exist in the living body. Of course the processes by which the body makes insulin are certainly quite different from those used in the test tube. And to start with, evidence has been acquired very recently, that insulin is not synthesised in the body as two separate chains but by a single chain, proinsulin, as discovered by Steiner in Chicago. Could I have the next slide. This is the way that the body synthesises insulin. A single chain governed by the usual processes of no doubt genetic processes that lead to a single chain. And then at a point in the pancreas, when it is transferred from the site at which it is synthesised to the storage cells, the islet cells of Langerhans. A proteolytic enzyme chips off this very long peptide fragment that connects the two chains. There is a sort of curios point about this if one looks at the molecule because one might think that this connecting peptide had to bring certain parts of the molecule near together, actually these parts are very close together, the actual beginning of the A chain and the end of the B chain in space. The beginning of the A chain is just down about here and the end of the B chain here in the molecule, only about two residues apart. So that one feels that the whole of this long peptide isn’t necessary only for bringing the chain structure, for encouraging the particular folding of the chain that we have in the insulin molecule. There are very interesting points about proinsulin. It does seem that if you break the disulphide bridges in proinsulin, the chains form again much better than in insulin and perhaps the long chain provides some kind of a template on which the molecule lies, which encourages the correct form of the insulin molecule. Or it provides some kind of protection for the molecule in its transport to the storage site. Now, the next slide shows a picture of the actual storage of insulin in the islet cells and now this is taken actually from rat islet cells by Dr. Howell in Sussex. And I think that you can see that the little insulin granules look sensibly as if they were crystalline. They look like little crystals and indeed in some animals you can see quite large crystals. But here they have the form, very much the same form as the crystals that we have been working on. If slices of these little crystals viewed in higher power in the electron microscope, in the next slide, you can see a repeat distance across them. In some views you get a view of lines of molecules. And if you look at the size of the molecule and the distance across of these lines, the distance is about 50 Å, which is sensibly the side of the rhombohedral unit, the distance apart of repeating hexamers. So it seems almost certain that in the actual insulin, the islet cells, the insulin is stored in the zinc containing hexamers. But now the problem is what is it doing. And here I have to throw this open really to the audience. The next slide just shows some of the different kinds of biological processes in which insulin has been implicated by a large number of experiments. And of these, of course the original ones that seemed most important were ones involving the metabolism of glucose, and particularly possibly the transport of glucose through membranes. We invite observations on this molecule at this point. But I might just add one more piece of evidence to it. We thought it would be interesting to look into the actual position at which the insulin molecule appeared to be able to change in different species without losing its effect. That is to say to map the species differences in insulin following the same kinds of ideas that Dr. Synge was talking about earlier. So in the next slide I have, not all but quite a number, you can see that many of the residues are allowed to change. And when you look at the ones that don’t change they’re generally - well, here the cysteine hasn’t changed, the leucine hasn’t changed, A19 tyrosine, A21 asparagine hasn’t changed. But a tremendous lot of changes have been observed. In the next slide we have just got a little map showing where the invariant residues are in the molecule as we see it, this is a single molecule. And they’re very largely ones that seem to be involved in pulling the molecule into its actual confirmation. The cysteine residues of course, but also two or three leucines that are involved in, as you might say, the heart of the molecule. And then this leaves us with just one or two on the outside, and A19 tyrosine is one particularly, and the asparagine which are also invariant so far. The last slide just gives you a picture of the molecule, this molecule, as you look at it here, but drawn from the actual electron density map, the actual positions taken out of the electron density map. And it’s really to give you an impression of what an approaching reagent would be seeing, you see this face full of detail and one asks oneself what parts of it are important for the actual biological reaction. Is it a particular region on this face or is it the whole organisation and is it the monomer, a single insulin molecule, as Sanger believes, or the dimer, as they believe in York, or the hexamer, as we sometimes believe ourselves, when we see the very beautifully organised object that it is, with pockets in it that might carry things about, you know. But this is still to be found out. And before I leave this subject, I should say two things. There are other crystalline modifications of insulin, very interesting ones, which are being worked on at the moment. The orthorhombic form, which contains no zinc in Columbia, by Dr. Barbara Low. The monoclinic form, which contains hexamers as its asymmetric unit by Michael Rossmann in Purdue. And at the end I should also say that in this particular research the sum total of the co-workers, the part played by the co-workers, the sum total was much greater than the part played by the Professor, to take your words from you, Professor Ruzicka, and that I should mention particularly those who carried through the last stages of the analysis, it had rather a long history. Margaret Adam and Tom Blundell from Oxford, Guy Dodson from New Zealand, Eleanor Dodson from Australia – they met and married in Oxford – and Vijayan from Bangalore - and I must say, we partly chose him because his name meant ‘victory’. Thank you.

Dorothy Crowfoot Hodgkin (1970)

Structure of Insulin

Dorothy Crowfoot Hodgkin (1970)

Structure of Insulin

Comment

This is the first talk, but not the last, given by Dorothy Crowfoot Hodgkin at the Lindau meetings. Six years after her Nobel Prize in Chemistry, she is still occupied with the crystal structure of insulin, a project she started in Oxford in 1934, more than 35 years earlier! She had then just spent two years with J.D. Bernal in Cambridge, learning the basics of crystallography of biological molecules. The choice of the special protein insulin was made because it plays such an important role in the body. In the story Hodgkin tells here, there are also many remarks as to the appearance and function of insulin in the body. But mostly, the story unravels some of the difficult technical steps involved in understanding the crystal structure of insulin. Since the lecture was given with many slides, this is not the right place to learn all the details, but the general story clearly comes through. One question treated is the molecular weight of the insulin molecule. Chemistry Nobel laureate The Svedberg had used his invention the ultracentrifuge and answered 36 000 units, but it turned out that he had measured particles of insulin, not molecules. Frederick Sanger, another chemist who also received the chemistry Nobel Prize, managed to determine the chemical structure of insulin by cracking it into its pieces. (He told his story at Lindau in 1960.) Insulin turned out to have molecular weight around 6 000 units, much smaller than Svedberg believed, but still big enough to give rise to many difficulties. One such was the need to insert heavy atoms in the crystals, a general method employed to get more information out of the thousands of X-ray diffraction pictures taken and inspected. At the National Portrait Gallery in London there is a wonderful painting of Dorothy Crowfoot Hodgkin working with her tables of crystallographic data using no less than four (!) hands.

Anders Bárány

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