"I see myself as a ribosome biochemist"

Mikel Valle: You're from India and your father was a biochemist, which is quite an unusual job. Did that influence your choice for science?

Venkatraman Ramakrishnan: Probably. I think I was exposed to science from a very early age. I used to go to see my father at work or go for a walk with him after work, so science was quite familiar for me. Moreover, scientists from different countries used to visit us and even stayed with us; eventually, my parents organised conferences with speakers from Europe or America… All this just gave me a flavour for the world of science. But my father really wanted me to be a doctor, a medical doctor, because he felt like any other parent, he wanted me to be safe.

Yes, I agree to some extent...

He didn't want me to go into science, and in fact I think he was kind of disappointed... maybe that's not the right word but he didn't like it. I made a deal with him: there was a national scholarship for basic science in India, and it was highly competitive, so I told him "if I get the scholarship, then you should not tell me what to do, and if I don't get it, then I'll do medicine". And I got the scholarship.

So you were lucky

I don't know if I was lucky. In the end, it all worked out.

During that time, in years 1960 and 1961, you went to Australia

Yes, my father was there as a sabbatical visitor granted by the Rockefeller Foundation. I was 8 and 9 then.

And later on you graduated in Physics. In the beginning you were not interested in Biology?

Actually, I was interested in Mathematics, but everyone told me there were no jobs in Mathematics, and of course this was before all the computer information revolution. I think they were right, there were fewer jobs. I also feel I was not that talented to be a real mathematician. So I chose Physics as the nearest alternative, a kind of a compromise, something practical but also mathematical.

In 1971 you were 19. Is this the usual age to graduate?

No, I skipped two grades at school, so I was two years ahead.

After graduation, you moved to Ohio directly?

Yes

There you did your PhD in Physics. What was the type of work you were doing?

I was a condensed matter theorist, so I was looking into phase transitions and ferroelectrics. I found I didn't have a good feel for the problem. I was just doing calculations. When you read about physicists like Feynman, you see that they have a deep physical insight into the problem, and only then they do the calculations, they have a sort of golden mind of what they want to understand, and I was sort of more blindly, saying let us use this approach to do this sort of thing. So I didn't like it very much, and part of it was the environment I was in and part of it was my own interest. And I became interested in lots of other things that were distracting when I was in graduate school so I have to mainly blame myself.

When you look at condensed matter theory, not a lot has come out in the last forty years, that is, something really groundbreaking in terms of understanding condensed matter physics. I think this is a problem for the whole field in Physics, fundamental problems are really difficult and they need new tools or new ways of thinking to attack them. In the meantime, everybody is calculating and it loses that excitement of making fundamental discoveries.

It doesn't look a pity to you to lose the future you could have. Just the feel itself.

Partly personal. I think to make a breakthrough now in Physics you have to be incredibly smart. Because the problems just stop, the people they feel they stop. But in Biology it is not like that, in Biology there are many fields to explore by ordinary people, you don't have to be a genius to make progress.

So you choose Biology directly as the only possibility you could consider.

Specially Molecular Biology was booming. In the mid 70's DNA sequencing was just beginning to be discovered, all this recombinant methodology was being developed... It was a very exciting time to enter the field.

And actually the contribution of the people from Physics was fundamental for that revolution, wasn't it?

Partly, yes. Sharp people like Francis Crick, Max Delbrück... they were in Physics before they switched.

So you went to the University again as a student.

Yes, that was a bit unusual, even in the US. I wrote to a number of Universities because I didn't want to be a postdoc, because I thought that if I moved from theoretical Physics to do a postdoc in some lab it could only be a lab that could use me for my Physics skills, and I would have a very narrow background. So I wrote to a number of places, and actually the chairman of Yale said they wouldn't take me as a student and told me to send my CV around to the Faculty. "If anyone wants to stop you for your postdoc, you could maybe do your transition that way" he said. And actually two people wrote to me - Don Engelman and Tom Steitz, but I turned them both down to go to graduate school, because I felt I wasn't ready for a postdoc.

So you wanted to keep control of the biologic part in order to be...

I wanted to have a broad background before I chose something, because otherwise I'd be going to a lab just because they accepted me, because I had a non traditional background and it may not be the most interesting thing for me. So the first year I really packed my whole schedule with courses: Genetics, Biochemistry, Cell Biology, all the standard things and the second year I took more courses and also worked in the membrane lab for doing research.

It sounds funny that Tom Steitz was one of the scientist who offered you a position

Yes, and when I moved for a postdoc I decided to write to one of the people who had written to me in Yale, but it was not Tom, it was Don Engelman and it was because I read an article that he and Peter Moore wrote in Scientific American on the ribosome using neutron scattering. I used to read Scientific American a lot. So I thought that if he wrote to me offering a postdoc, he might be interested in me by then. I was interested in membranes, and Engelman is a membrane protein guy, so I wrote him and he answered he did not have any membrane protein positions but that there was a postdoc position on a ribosome project, and if I were interested, he would tell Peter Moore about it. So I accepted, and Peter came to San Diego for a meeting and after the talk he offered me the job.

So it was your first contact with the ribosome.

That's right.

Was it immediately interesting for you?

Yes, it was interesting. I read that Scientific American article, and by that time I knew why the ribosome was important and so on. It seemed just a perfect problem for a Physics trained person to get started because of its large molecular machine, so you could think of it from the Physics point of view but also from the biological fundamental so you had to understand all that. It was a challenging problem and also I felt it was a problem that would last for a very long time, not just to do a postdoc and then find something else to do.

You could anticipate then that it could be something you could do for a long time.

Yes, a long career.

Did you start immediately doing X-ray crystallography?

No, actually I didn't do X-ray crystallography until I got tenure (ie my job was made permanent). My training was in biophysical methods: neutron scattering, ultracentrifugation, things like that, so I was applying these approaches to both chromatin and the ribosome. And then Brookhaven had a tenure system so when they asked me what I would do if I got tenure, I said I was limited by my techniques and I had to learn how do to high resolution structures to make a link with chemistry and function. So I wanted to change my emphasis. I decided to go on sabbatical to do it. So this was really strange to decide to stop your work for which you were given ten year, but they were very broadminded.

Was Peter Moore doing X-ray crystallography by then?

No, it was much later. The Yale group started only one year before us, in 1995. I actually wanted to start in 1995 but I had just moved to Utah, so it took me a year to set up my lab and get everything going. So I was delayed a little bit.

So crystallography started in Utah?

So I got tenure, and this was in the late '80s, so what I did was go to the first Cold Spring Harbour crystallography course and this was in October 1988. Actually, very famous people were my teachers like Hans Deisenhofer who gained the Nobel prize for the reaction centres the day after the course finished. Then I went to sabbatical at the MRC (from 1991 to 1992). By that time, I already collected two data sets: one on a ribosomal protein S5 and one on a histone, which is involved in chromatin structure H5. So I took the datasets with me but I didn't know how to solve structures so at MRC I learnt how to take your data and look at maps and solve structures and so on. So by the end of the year I had two papers, and both were published in Nature, so that was a successful sabbatical. And then when I came back for the next few years we were working on structures of individual ribosomal proteins by we and Steve White who actually started that project from Berlin. In the meantime, I applied all these cloning methods because the T7 system was developed at Brookhaven, so I was slightly ahead of most structural biologists. The people who developed it were right at our next door so we were able to very quickly overproduce these ribosomal proteins, and then we started solving their structures.

So the strategy was solving proteins, maybe proteins with small pieces of RNA?

That was the next stage, solving the L11 – RNA complex, because the L11-RNA was the binding site for antibiotic thiostrepton and for elongation factors like EFG which help the tRNA and mRNA move through the ribosome. So we thought it was a very interesting complex that was very well characterised. And so we ended up solving its structure.

But then you realised that you didn't learn a lot from anyone of those proteins. Actually the first one was interesting and it was published in Nature, but each one became less and less interesting, and then the protein-RNA complex again was a big story and it was published in Cell but then again you realised it didn't tell you that much about how they interact with the factor and how the ribosome works. And if you consider all the protein RNA fragments, it would need an enormous amount of work, each of those would be around five years of work. And I thought the time was right to attack the whole problem. Since there were already good crystals of the 50S I didn't want to do that, so I thought that maybe I could improve the crystals of the 30S subunit, which at the time were around 8-10 Ångströms resolution. And I had some ideas which turned out that still hadn't work, binding IF3 with the 30S subunit and maybe locking it or something, getting better crystals. But before that we tried just to crystallize the 30S by itself, to see if we could reproduce the poorly diffracting crystals and that ended up diffracting well when we payed careful attention to the biochemistry.

What was the key point to get good crystals in a general way?

By 1995, when I went to Utah, I tried to attack the whole problem. After one year for setting up, then start up and recruitment of two students in the lab, things started to progressively get better. Then we collected our initial crystals and ran a gel on them. We realised that when you make 30S subunits, they contain a mixture of subunits with a protein S1 – involved in some activities but not required for most of what the ribosome does – and those which don't have it. The protein is known to be loosely bound and the crystals are of ribosomes that lack that protein. So we very systematically removed that protein and I won't say that was what gave us high resolution crystals but it allowed us to reproducibly grow large crystals that were diffractive. That was one breakthrough. The other breakthrough was in thinking about how to face the crystallographic problem, because people like Ada Yonath had 50S crystals for a long time and were not doing much progress in getting a believable structure at that point. So it was clear new ideas were needed. And the Yale group had a certain set of ideas which they started with. And my idea was really to use anomalous scattering to get phases. That idea eventually was used by the Yale group, by us and by Jamie Cate . It was not a profound idea but it was the correct idea. But in the meantime, the Yale group had figured out how to get starting low-resolution phases from heavy-atom clusters. I don't think this is absolutely necessary (because there are now good methods that will help you locate the anomalous scatterers directly), but it certainly made it much easier to get started, the way that they did it.

So success, and then the structures came out in 2000 with fundamental insights in decoding, and now there are a lot of crystal structures. If you already solved the structure of the ribosome, who cares for the next one?

It's becoming harder and harder to get students and postdocs. The trophy value has gone. But if you're really interested in mechanisms, you have to think of the ribosome as a large machine, like a complicated car – a Ferrari or a Porsche. Let's say you're a Martian and you come into the Earth. Initially, all you know is that this thing takes gasoline, emits CO2 and the wheels go round and it moves. But if you open up the hood and look into the machine, you realise it is a really complex machine. If you only see the machine when it is not working, you may have some ideas of how it works, but you don't really know how it works. And what you have to do is take snapshots of the machine at different points as its working and effectively make a movie of this machine. That will give you a much better idea, it won't tell you the whole story. The whole story won't come from making up pictures, it will come from Biochemistry, molecular dynamics calculations and so on. But the more pictures you have, the better understanding you get. And of course, people like you can take a lot of frames in the movie, but the advantage of crystallography is that you can still see directly where the chemical groups interact, the local changes where they're involved and things like that. High resolution crystallography has always added a level of understanding for release factors, EF-Tu structures, etc. That's the idea, to get a chemical snapshot of the machine.

Have you ever changed a strategy to get crystals from your complex because of a cryo-EM low resolution map?

No, Tom Steitz said "Life is all about hydrogen bonds". If you want to narrow down to chemistry, then you have to do that. Cryo-EM is tremendously valuable, and specially in combining crystal structures with complexes. Many of these complexes are not going to crystallise, because they are too dynamic or things like that, or materials with limited stability. So I think there is a terrific room for both. In fact I think of doing a sabbatical in cryo-EM if I can learn that at some point. I think if you ask a geneticists or a biochemist which one would they have, there's no question that they will want crystal structure.

So at the end you are now working in Biochemistry, or Molecular Biology, since they go together?

I see myself as a ribosome biochemist. You shouldn't define people by technique, because if they would to call me a crystallographer rather than a ribosome biochemist, then I would say "you´re are gel biologists", because what some do is run gels. They're using gels as a technique, I'm using X-rays as a technique.

Your father worked as a biochemist and now you are doing something similar...

Yes, but using more mathematics and physics.