…I’ll talk a bit more about inspiration.
After yesterday I think that I really have to show you what I called yesterday the evidence.
How this project started and continued.
Maybe it is as important as the result.
So let’s climb the Everest beyond the Everest.
I think that you all know that the Ribosome is the last chain,
the last member in the chain of translating the genetic code into proteins.
The genetic code is being expressed in messager RNA which is very similar to DNA
but can exist as a single strand therefore it can direct the information and it is available for translation.
So ribosomes are universal.
Each ribosome can translate every messenger, every sequence in a similar fashion.
There is a huge number of ribosomes that function in each cell.
Mammalian cells can contain millions of ribosomes.
This is a piece of information I didn’t know when I started.
Even bacteria can reach in the low period they can reach 100,000 ribosomes functioning together.
In my opinion, I'm a chemist or at least I studied chemistry, ribosomes are amazing molecular machines.
They act continuously, and they can make between 15 to 40 peptide bonds in one second.
I needed eight hours in the lab.
You can imagine what would happen to your life if (laughing).
I also needed 100°C and PH of 2.
And the ribosomes can do it in every cell under almost all conditions known to support life.
They also hardly make mistakes.
The numbers in the literature until recently was 1:1,000,000.
Today people speak also about 1:10,000.
In my opinion both are admirable.
The main constellation is an L-shaped double strand with an anti-codon loop that can make codon,
anti-codon interactions here like (inaudible 00:02:25).
They carry their amino acid far away from here, it’s about 70 angstrom always in all cells, which is coordinated to this one.
So the messenger RNA is attracted and bound to the small ribosomal sub-unit,
all ribosomes are made of two sub-units no matter from which cell they come.
And they exist like two separated sub-units until they start functioning.
So the cell has two different sub-units, two separated sub-units.
They associate with each other when they have to function.
The functioning is first to attract the messenger then to design the first initial codon to be translated
into the translating decoding site which is called the P site tRNA site.
tRNA binds to there by codon/anti-codon interactions and the proof reading machinery
that everything is fine is also here in the small subunit.
This is why we call the small subunit the brain of the ribosome.
The hands that make the bond are in the large ribosomal subunit and this is universal and no matter which cell we are looking at.
So what I just told you is shown here, the small subunit has a narrow part here.
In the past also today people like to call this the head and the body and the neck of the small subunit.
Messenger wraps around it.
The first site to be populated is called P peptide transfer, this is tRNA.
Symbolising the tRNA with the amino acid.
There is another side that will be occupied afterwards call the A-site tRNA
where the tRNA where amino acid will come, amino acid related tRNA.
And there is an exit side from where the tRNA will go out afterwards.
So once we have this all made, small subunit, messenger and tRNA and initiation factors which I don’t show here,
non-ribosomal initiation factors, the large subunit can come.
The surface complementary is very good but not perfect.
So in order to have it perfect first of all inter-subunit bridges are being made like those.
Bi-conformational changes mainly of the large subunit but not only.
There are thirteen of those and I symbolise them in three triangles.
When everything is fine the P-site tRNA with the amino acid is positioned
so the amino acid is on the entrance of a very long tunnel that spans the large subunit.
For which the newly born protein will progress and exit.
So now everything is ready for the next journey.
It will go now more or less sideways to this position and make a peptide bond here,
which is the first bond for the newly born protein like that.
So now we have a D peptide beginning of the newly born protein of the nascent chain.
The first tRNA is now empty, it can go out and a new one can come in.
These things happen more or less the same time but I'm just a human being I couldn't draw them at the same time.
So that’s the story and it will continue, the translocation from A to B and to E is mainly in this direction.
So many ribosomes act together, you can see it here, I must say that I am amazed by this electron microscope grid.
Every time I look at it again it's from the ‘70s, I don’t even know who did it.
I found it in the literature without reference.
It shows many ribosomes, these black things, black bodies, that are bound to the same messenger RNA act continuously.
You can see that each of these black things have two subunits, you can see here small large, small large, small large,
small large and so on.
You can see that they read the same messenger and the protein comes from the other side.
The only thing that is not exactly as it happens in the cell is that the protein comes out flat.
Within the cell you surely know it falls immediately when it comes out of the ribosome, either alone or with chaperones,
with the aid of chaperones.
So that’s the story.
When we got the first structures we made a movie according to them.
So the movie that you will see now was made by art students, based on our coordinates together with us
with interpolation between our structure and others as it was known in 2002.
So messenger comes to the small subunit, it waits for it and pay attention, now, now it comes and it took it,
it’s a big conformation of change.
These ones is our initiative factors, the blue ones, initiation factor three and two.
The two brought the first tRNA and now we have an initiation complex,
the large subunit can come and make bridges by conformational changes.
And we have an active ribosome, that will accommodate the tRNAs that are brought by other factors, elongation factors on one side.
And help the empty ones coming out on the other side, ribosome is really working.
Peptide bond is being made here, the decoding is there.
Now we take away the large subunits and you see something that I want to mention.
The motion is more or less sideways.
But here on the other side of the tRNA there is a conformational change.
I just said I won’t be able to talk about it much today, but at least pay attention.
So their proxies continues until there is a stop codon.
And then the tRNAs being replaced by factors, either release factors or recycling factors.
This gold thing that will come in and separate between the two subunits.
The tRNAs can go home, the protein can fold, the two subunits can wait for the next job to do.
So what you saw in the movie was not very clear, we know now the position of every atom in the ribosome.
Molecular weight of the bacteria ribosomes is 2.5m.
What you see here rotating is bacterial small subunit.
They are more or less the same in all bacteria.
Changes are minute, the sediment with the sedimentation coefficient of 30 so we go 30S.
Total molecular weight of the small subunit is 0.85 Mega Dalton.
And what you see here rotating is the complex, the RNA, the ribosomal RNA is in grey or silver.
And in the small subunit of bacteria it contains about 1,600 nucleotides.
The curly things, the coloured curly things mainly on the surface are ribosomal proteins.
Each of them is now shown in a different colour.
There are up to 21 different proteins in this small subunit, it means between 20 ~ 21.
And the task of the small subunit to remind you is decoding of the genetic code.
The large subunit its task is to make the peptide bond, to make sure that it’s being elongated, the nascent chain is elongated.
It means it’s a polymerase and to protect the newly born protein in the tunnel that I showed earlier.
Sedimentation 50 S.
Total molecular weight is 1.5 millions in bacteria.
Two chains of RNA total 3,000 nucleotides, one of them very long, 2,800 and the other the rest.
And up to 34 different proteins.
When we look at the surface that will come together in the assembled ribosome like that we see that the decoding
here in the small subunit and peptide bond formation here
and you can see here how they are connected by the tRNA decoding here and peptide bond here, the amino acid is bond here.
We can see that the region where things are happening, the two regions here and here are made of RNA, not proteins.
Only silver, which tells us that the ribosome is actually a ribozyme.
It’s an RNA machine.
And this is in good correlation with the ribosome composition.
In all ribosomes except in mitochondria the ratio of RNA to proteins is 2:1 it means twice as much RNA.
Even in mitochondria where part of the RNA is changed by proteins the active sites are preserved and they are RNA made.
And this was suggested by Francis Crick back in the ‘60s was not easily digested by scientists
who thought that making a bone its enzymatic reaction and enzymes are known to be proteins.
Well, Francis Crick was right!
What does it show us?
What does it hint at?
It hints that first of all usually RNA machine, ribosomes are lousy proteins, not efficient, lousy enzymes.
Not efficient enzymes.
There are not many known today and all of them are rather slow and sometimes not very selective.
The ribosome as I said earlier is an extremely efficient machine.
So nature found a way to improve the ribosomes, to improve the RNA machine and make it efficient.
Nature can find ways when it’s needed.
Second it shows that RNA existed before proteins.
I want to talk about it for a second.
We think that within this complex ribosome we identified a little, a little piece.
It looks here large because we zoom on it but it’s actually not more than 3 or 4% of the whole RNA,
of the ribosome depending if it’s bacteria or higher organisms.
This region can make the peptide bond and it is in our opinion a remnant of the prebiotic machinery for bonding.
That is still functioning today in the modern ribosome.
We are very excited about this finding and we are trying now to understand it better and to prove it.
On what do we base our thinking?
I cannot go into the whole thing now I just tell you the most important two facts.
First of all it can make peptide bonds, the whole machinery is there.
Second, it has an incredible conservation.
So out evolution 98% conservation.
The last is that when we look only at that machine from a chemical point of view it is stable,
it can make the bond right here and it has orientation that can support itself.
So that’s what we call the pocket.
Unfortunately I can’t talk about it today.
But, from this we studied something, or we can suggest something.
It’s a question that the world is busy, what was first the chicken or the egg?
The DNA, the genetic code or the proteins?
Or the machines of life.
Well in our opinion first was the ribosome, not the genetic code, neither the proteins.
Okay ribosome is the target for antibiotics, the natural antibiotics are weapons
that bacteria from one type uses for eliminating other types.
And because the ribosomes are so important producing the proteins they are target for many antibiotics that are of clinical use.
Over 40% of the useful antibiotics target protein biosynthesis.
We like to compare between the ribosome and antibiotics to David and Goliath.
David this little kid had a very small stone and he had to eliminate this huge Goliath that was mainly covered and protected.
So he aimed at the forehead because the forehead was exposed and important.
He didn’t aim at the hands for instance.
He was successful.
Because he touched a function, a very important function of sight.
The reason that we compare or we like to discuss it like that is the molecular weights.
Antibiotics are less than a 1,000 ribosome, bacterial ribosome which are the ribosomes that we want to hamper, they are 2.5m.
How does it happen exactly like the stone of David?
The antibiotics hit the ribosome at its functional sites.
It disturbs the function.
So here are some ribosomal antibiotics that are bound in their binding position to the small subunit
where the messenger binds or where the tRNA has to be decoded.
All those that allow the motion that is associated with it, and in the large subunit again those that allow the motion,
you remember in the movie there were hands going in and out.
These disturb the motion or in the active site or at the tunnel for which the proteins go.
I want to discuss only a few of them.
First what you see rotating is the large subunit RNA in blue and all proteins in green.
The three colour things inside, the magenta is Chloramphenical that binds to the A-site.
To the lower part of the A-site.
In yellow is Clindamycin that disturbs the creation of the peptide bond.
And in red is Erythromycin that binds to the tunnel.
Erythromycin is the first antibiotic that targeted ribosome that came into use.
And I want to show where it binds.
So first let’s look at the tunnel.
Here is the large subunit, with A and P tRNAs in it, a bond will be made here.
And the tunnel is highlighted here but modelled polyaniline.
So the tunnel has an uneven shape, and near the constriction the narrowest part is the binding point of erythromycin
and the family that came afterwards is called macrolides.
You can see here what we mean by blocking the tunnel.
So protein will be made here, what you see here is a section for the ribosome like we'd like to do with apples.
Peptide bond is being made here, protein will go through the tunnel if it’s not stopped by erythromycin.
Looking into the tunnel from top this is the large subunit, entrance to the tunnel a little bit lower,
erythromycin blocks most of it so protein cannot go through.
Macrolides are made of a ring, the normal macrolide erythromycin 14 members and 2 sugars.
Erythromycin was found to be a very, very successful and very useful antibiotic
when it came into use in the last century, in the middle.
But not very stable in acid solutions which is the stomach.
And companies modified it to make the stability higher in two positions.
I want to show you want happened.
The first is looking at entrance to the tunnel, what you see here is the whole RNA of the large subunit.
Because the antibiotic macrolides bind only to RNAs, I can show just RNA.
Entrance to the tunnel I zoom it erythromycin binding way.
To take several more binding modes but all of them are blocking the tunnel with more or less the same way,
it shows that there is flexibility even in binding depending on how the experiment was done.
However, the binding, the anchors are the same.
Addition of clarithromycin better blocker and more stable, addition of a longer arm is roxithromycin that better blocks
and is more stable in the tunnel because it has more anchors.
And maybe this provides to my understanding only the first time for antibiotics the structural basis for dosing.
So in many cases 150mg of roxithromycin twice daily has the same effect as 500mg of erythromycin four times a day.
So it’s 1:6, it’s very impressive.
So I already say that all antibiotics bind to ribosomal functional sites.
I also say ribosomal functional sites are highly conserved.
Mandatory for clinical use is the distinction between the pathogen and the patient, we want to kill the pathogen not the patient.
So if we target ribosomes without distinction it’s a problem.
How do the antibiotics differentiate between patients and pathogen, by subtle differences, I want to show you one.
So here is section through the tunnel, these are the tunnel walls.
You cut it like that.
And you already saw that it has an uneven shape.
So in grey is the tunnel.
Inside here are all types of structures of antibiotics from the macrolide family,
erythromycin family that block the tunnel perfect, almost perfect but always very good.
And only one nucleotide is being highlighted A2058 nomenclature according to e-coli.
A2058 is the name of the game, it’s adenine in new bacteria.
Adenine, erythromycin very high chemical affinity here.
All pathogens are new bacteria.
So all pathogen can bind this way.
Have a look here, this is us, all higher animals.
G instead of A in this position.
But when there is a G here, this is the only difference
when there is a G here there is rejection based on two short contacts and no binding.
That’s all.
So I showed you how the differentiation is made, now I have to talk about the problem, which is resistance.
And resistance is a very, very acute problem in modern medicine.
How do the pathogens acquire resistance?
Prominent way is to hit the anchors.
Like here, you remember 2058 now you see it from the other side.
An erythromycin these are good contacts and this is the basis for selectivity.
It’s also the basis for resistance.
So the bacteria, the pathogens can change their own genome from A to G, or they can make a post translational modification,
ERM modification which is methylation in this position.
Most mechanisms are really involving the modification of such anchors
but there are some that are mediated by remote changes and I want to show you an example.
So again you see here the tunnel.
You can see it now in the whole ribosome, it is here.
And you can see that the tunnel walls are made mainly of RNA
but there are two proteins that their tips are getting into the tunnel wall, here you can see here.
Macrolide binding site is more or less here, those are below it.
Yet, it was found already in the ‘70s that minute mutations in these two proteins L22 and L4 can acquire resistance
to erythromycin although L22 and L4 are not part of the binding site.
And although the resistant mutants bind erythromycin.
So how does it happen?
We really wanted to know it, in collaboration with Dr. Janice we got this mutant in the ribosomes that we can crystallise,
we have crystals now for the mutants
and what we found is that this minute changes did not really change much in the protein position.
But triggered a whole chain of changes in the RNA walls.
So that the tunnel can now accommodate erythromycin and the newly born protein.
So this is a way that we didn’t know about until recently.
What do people, companies do in order to combat resistance?
I think that always it will be only partially combat, maybe controlling it.
Because bacteria wants to live, but what do we do?
Sometimes there are new compounds with additional anchors or synergies that have two compounds together.
For instance additional anchors or higher flexibility is shown in azithromycin by the addition of one atom.
Just compare this to erythromycin almost the same chemistry.
And it’s one of the most successful antibiotics, it also works against resistant strains.
But I was worried when I heard about it, that the Croatian group developed it.
Because if it binds resistant strains that have G instead of A it will also bind to the patient.
So what did we get?
Luckily I was wrong, and this you can see by comparing the binding of azithromycin to a model for pathogens,
the green, and to a model for patients the H.
I cannot talk much about it here and now but you can see the binding happens.
But sometimes it’s along and sometimes it’s across.
This shows that binding, 2058 is only showing if there is binding but not how.
Two compounds, there is one commercially available drug called Synercid, it has two compounds,
one in the active side and one in the tunnel.
You want to see how it binds, here is the tunnel wall one and second they glue each other and they block the tunnel.
Make me a bit happier and we looked for another one.
So Synercid is what I showed you, this is another one.
Two compounds, pair of compounds produced by one drug that goes in Sri Lanka, this is why it’s called Lankacidin and Lankamycin.
We found that both of them bind together, that the interaction between them is very strong
and they introduce also conformational change.
So we have some hopes about that.
Also we found that Lankamycin which is binding to Lankacidin is very similar to erythromycin,
yet erythromycin is a competitor whereas Lankamycin is a binder.
And this gives us a tool to make the coupling better.
So a movie that may work of how some of the antibiotics that we know today work, Edeine doesn’t let messenger binding.
The next one will be Tetracycline that doesn’t let a-site tRNA binding by occupying the space, the position here.
The third one is erythromycin that we discussed.
This is erythromycin, the last one is clindamycin that doesn’t let the bond being made.
So a minute about inspiration.
We are using crystallography I think that you all know that by using x-ray crystallography
we can see details that we cannot see otherwise.
The idea is we have crystals, we get diffractions in all directions,
we collect it and we make if we know how to collect them together and face them we do it correctly,
we merge them correctly we get electron density maps
and since x-rays are interacting with the electrons we know from there where the atoms are.
And I really don’t go into details just see what I mean by diffraction.
So here there is a crystal beam comes in, and is diffracted to all types of directions with all types of intensities.
But we need crystals.
And crystals are entities that have perfect order on three dimensions or almost perfect order.
So in salt, sodium chloride is no problem.
In the middle of last century people started to get crystals even of proteins
and a 6th grader could repeat it so it’s possible, this is lysozyme.
That was crystallised even by a child now.
But those ribosomes that are so complicated and you saw that they are very flexible.
I didn’t say that they deteriorate fast, there was a big question mark when we started actually the question mark was negative.
It cannot be done.
And these are the reasons that I told you earlier, the most important one in my idea is the marked tendency to deteriorate.
They deteriorate very quickly within one to four days in the test tube or in the body.
So why did I being young, almost your age, why did I start it?
Because I had a chance to read, even not only scientific, I read about the delegation that went to the North Pole
and looked at the metabolism of the sleeping bears.
And they found that while they were sleeping the ribosomes are orderly packed inside their membranes.
ribosomes can be orderly packed.
Every bear every winter, it’s not an accident.
Why do they do this?
This is what I thought because I thought this is the way to keep a large pool of active ribosomes
because when they get up they want to do things they didn’t do when they were sleeping and they need proteins.
So that more or less what you can see here.
I was not the only one that read it, many other groups read it for instance people used this finding
in order to make crystals or mono-layers actually on membranes.
You know what they did?
They took fertilised eggs they put them in the fridge and in three minutes they had this beautiful order.
So people thought that the organisation is because of cold, because of cooling, I thought that it’s general stress
and it was shown later that also bad diet does it.
But anyway this happens in the cell and all what we wanted to do is to make it in the test tube and use ribosomes from bacteria.
Not from bears and not from fertilised eggs.
So the solution was to crystallise ribosomes from bacteria that live under harsh conditions.
And to use procedure that extend the life a little bit, I was lucky, I found a one like this.
I want to show you harsh conditions for instance the Dead Sea, the lowest point in the world.
It’s in Israel, you can see it from top, you can see it closer.
These are salt crystals that come out of the very saturated sea.
But even on them bacteria grow.
You see here some colonies that grow on the salt, you can see it even better here.
The level of water here is much lower, it’s September.
This was the level of water in January about 70cm, they still grow.
So we found this could be good ones or these that grow in sewages off atomic virus,
deinococcus radiodurans that can grow also on dust in hunger, heat, cold because only quarter of it is functional
and the other four are packing their DNA and will come into action when one quarter has to be repaired.
They also have all the repair mechanisms of the known today.
And to use the procedures as I said earlier.
Using these procedures we could see even after a day evidence for organisation.
This is an electron micrograph of a drop that we put for crystallisation.
You see here order.
So even we could do it in the lab.
So yesterday we talked about persistance and belief.
I say faint but solid evidence were given all the way through.
I want to show you also only a few of them.
So the curiosity was the reason but the evidence was the driving.
We wanted to get this picture, in the beginning we got that, and this was for me good enough
because it had some evidence for high resolution.
And when we cut it, it was very ordered.
We looked at it, you see the benefit of working on very large particles is that you can see them in an electron microscope.
So we could see the order inside.
We needed twenty-five different conditions but it was not difficult it took only four months.
Because we devised a way, a matrix to watch it and when we found the region for instance the pH region
where we started to see the difference between solubitiy and sedimentation
we transferred it to capillaries and look how large the crystals became.
Were not still good but we developed.
And look how nice they were ordered inside.
So I don’t want to tell you about all the types of crystals that you can see from the inside,
I will show you in a minute from the outside.
We went from this to this, you remember what we desired, I showed in the beginning.
So this was our desire.
This is the story.
In four years after we started we had a few stops first time, still far but much more than in the first days.
And then we got really good crystals, many good crystals that could be exposed.
We went to synchrotron to measure, these are facilities where particles are being accelerated
and we see it on the tangential in stations and measure.
And we found six years after we started we had good crystals, very, very fantastic diffraction.
But after 0.1 of a second it disappeared.
The decay was very large and then we failed here comes the Everest.
We climbed an Everest, we have good crystals, fantastic but only there we found the way still very far,
the real Everest is still in front of us.
So we could stop then, we did not.
We thought why is there damage, I can’t go into detail but the main thing is damage happens by x-rays heating peptide bonds
or any bonds and we can only try to minimise the progression.
We did it by cryo temperature, look at my face when the experiment started.
In Stanford 1986, I was not very hopeful.
But this was the breakthrough.
And today with informants it became routine in the whole world and today almost all the structures are measured this way.
So we have wonderful diffraction and the whole world has many more structures.
So this is when we introduced cryo-crystallography together with some other developments for phasing and detectors.
Please multiply this by thousand and see how many structures are now in the world.
PDB, is protein databank deposition, each of them is a structure.
So the thanks and I said yesterday that there were only a few people that could grasp the potential of this work.
They didn’t really believe like I, they still doubted but they suggested it to the Weizmann Institute President
and they let me work for a long time on a project that was considered maybe or may be not.
So Sir John Kendrew, Chris Anfinsen and Alex Rich and the president Michel Sela and Haim Harari.
The whole work started as I said before in Max Planck in Berlin together with Dr. Wittmann
that was crazy about knowing the structure of the ribosomes.
He also helped us to have a work in Hamburg near the synchrotron.
He died rather early and was replaced by Franceschi and Fucini.
Now because we had two research groups at the same time
I am thanking for the young members there for their devotion and determination.
In good and in more than this bad times.
You can see the Hamburg group, with their angels, our technicians and looking for better bacteria in the Dead Sea.
And you can see the Israeli group that is still functioning, it’s run by Dr. Anat Bashan, she is the senior scientist.
I can’t tell you what everybody did but everybody contributed, what you see here is the collection of the last three years.
But I want you to look at Tamara, Tamara Auerbach she came to us thirteen years ago for ten weeks, she’s still there.
She came as a bachelor, she’s still, she now has three kids.
But the day I took a picture and this is the important thing, she had her birthday and she baked a cake.
And this is her cake which shows that for my group cake ribosomes are considered sweet.
I also want to thank my family that supported me for the whole time, it’s the whole family
but I want to focus on my granddaughter.
And the reason I'm doing it is for you young female that hesitate whether it’s possible to do science and have a family.
Have a look, you know what she’s saying, this is a speech she gave without telling me in Paris.
As you know she’s very busy, she means me, but she always finds time for me.
And at age of five she invited me to her kindergarten to explain what ribosomes are.
At the age of ten she gave me the most important prize in my life: The grandma of the year is Ada Yonath.
There is no year, and she told me I have to reprove myself every year which means it’s a loving but demanding family.
When I fail she will take it off.
Thank you.