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Electron microscope work and crystallising the histone octamer


Crystallising the nucleosome
Aaron Klug Scientist
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Roger then, I think, went off to Harvard and later to Stanford and we set about trying to crystallise the nucleosome, a single nucleosome without H1. H1 is the fifth histone, which we later showed with Thoma and Koller, it's absolutely essential for making a regular packed helix. That was a paper in '79, the paper got rejected by I don't know how many different referees but it turned out to be right in the end. How many different journals and referees... because they thought that a linear array would be enough, the answer... it really was at the basis of that. So the...

[Q] Len Lutter.

Oh, Len Lutter. So then we recruited Len Lutter and the idea was to try to find out what the nucleosome was, so he then... Lutter was an American Post Doc, a very skilful, very skilled at the bench and he began doing nucleosome preparations of different kinds. And he found, that if you added microchoccal nuclease then you could get a ladder, just as you went down in the one, two, three, four experiment, all the way down to single nucleosomes. If you went on digesting you found that the 200 base pair b, and turned into 160 base pair b, and so clearly something had happened. And during that time, I think I've forgotten which point we showed that, the H1, if there was H1 present, fell off. And there was H1 present because we did this from... originally from calf thymus chromatin, but I think that we got it from beef kidney in the end. Some people tell you, you can get better preparations from beef kidney, was it you who use to go to the abattoir to collect it?

[Q] No, no.

They used to have to...somebody... You had to tip the slaughterman to give you the kidneys.

[Q] Yes.

That was a good, useful source. And so we, so the... Ray Brown and Daniela Rhodes crystallised the nucleosome. It was quite an interesting crystallisation because it, if you actually started adding magnesium it precipitated. And they were trying to do things with manganese as well which had the same behaviour, and then Ray Brown and Daniela Rhodes, one day announced he's got crystals, but the crystals were at 40 millimolar magnesium; And I couldn't believe it. And then I realised what was happening, and I go back to insulin... this is not a digression this was actually part of the reasoning. You know, if you actually, insulin picks up zinc, this is the work of Dorothy Hodgkin, who worked out the structure, there's two zinc, there's four zinc and six zinc. And the different zinc they formed different... it's well known in organic chemistry they formed different... higher aggregates. And the... so it was clear that the... you could make six insulin because they were an aggregate of insulin molecules. And I realised that this must be an aggregate at 40 millimolar of the... so the... so what was happening before... so we managed to grow good crystals because if you... if you, so the... the low amounts of manganese they precipitate out and they don't give good crystals at all, no crystals at all. If you go on adding manganese, they salt in as the physical chemists say, so we actually determined the boundary at about 40 millimolar. It turned out to be dependent upon the exact length of the DNA that you chopped up. Normally it was about 100 and 160 turned out to be only a rough measure and the best crystals came with 147 bases of DNA, and these were crystallised.

Born in Lithuania, Aaron Klug (1926-2018) was a British chemist and biophysicist. He was awarded the Nobel Prize in Chemistry in 1982 for developments in electron microscopy and his work on complexes of nucleic acids and proteins. He studied crystallography at the University of Cape Town before moving to England, completing his doctorate in 1953 at Trinity College, Cambridge. In 1981, he was awarded the Louisa Gross Horwitz Prize from Columbia University. His long and influential career led to a knighthood in 1988. He was also elected President of the Royal Society, and served there from 1995-2000.

Listeners: John Finch Ken Holmes

John Finch is a retired member of staff of the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK. He began research as a PhD student of Rosalind Franklin's at Birkbeck College, London in 1955 studying the structure of small viruses by x-ray diffraction. He came to Cambridge as part of Aaron Klug's team in 1962 and has continued with the structural study of viruses and other nucleoproteins such as chromatin, using both x-rays and electron microscopy.

Kenneth Holmes was born in London in 1934 and attended schools in Chiswick. He obtained his BA at St Johns College, Cambridge. He obtained his PhD at Birkbeck College, London working on the structure of tobacco mosaic virus with Rosalind Franklin and Aaron Klug. After a post-doc at Childrens' Hospital, Boston, where he started to work on muscle structure, he joined to the newly opened Laboratory of Molecular Biology in Cambridge where he stayed for six years. He worked with Aaron Klug on virus structure and with Hugh Huxley on muscle. He then moved to Heidelberg to open the Department of Biophysics at the Max Planck Institute for Medical Research where he remained as director until his retirement. During this time he completed the structure of tobacco mosaic virus and solved the structures of a number of protein molecules including the structure of the muscle protein actin and the actin filament. Recently he has worked on the molecular mechanism of muscle contraction. He also initiated the use of synchrotron radiation as a source for X-ray diffraction and founded the EMBL outstation at DESY Hamburg. He was elected to the Royal Society in 1981 and is a member of a number of scientific academies.

Tags: Ray Brown, Daniela Rhodes, Dorothy Hodgkin

Duration: 4 minutes, 14 seconds

Date story recorded: July 2005

Date story went live: 24 January 2008