Count Bernadotte, ladies and gentlemen, I would first like to take the opportunity

to thank you all, and Count Bernadotte particularly, for the invitation which brings us here. This is unfortunately the first time that I have been able to attend one of these occasions, and I realize how much I have missed.

I would only like to point out to Count Bernadotte how widely my subject extends. A few years ago, the young people who were working in my physics laboratory, two of them

shared the Nobel Prize for chemistry, and two of them shared the Nobel Prize for medicine. There is therefore, Count Bernadotte, no reason that I can see why you should not invite me every year to the course if you like to do so.

As our producer has said, the story I want to tell you is the history of a research which lasted over 25 years.

I have always been interested in trying to apply X-ray analysis to more and more complicated bodies. And this story is the analysis of the most complicated bodies

which have yet been successfully investigated, the protein structures. When Perutz and I started on this 25 years ago, success seemed almost impossible. They were so complicated.

But the prize, the reward for getting them out, was a dazzling one, and we felt we must have a try. Now, perhaps I may just outline the problem. The protein molecules are large molecules

which play a part in the processes of life. Hemoglobin, the one with which we started, is a molecule containing 10,000 atoms. It has the special function in the body of conveying oxygen

from the lungs to all parts of our body and then taking back the carbon dioxide again to be given out in the lungs. When we started this investigation, the hemoglobin which we chose is a molecule

containing 10,000 atoms. So far, the most complex structures which had been investigated were those done by Dorothy Hodgkin, in particularly the structure of vitamin B12,

which contains 181 atoms. The difficulty of getting out a structure goes up as a high power of the number of atoms in it. So we were trying to do a molecule with 10,000 atoms when the best so far had 180.

May I remind you of the nature of X-ray analysis? X-rays fall on a crystal structure composed of the molecules which you are examining.

You notice how strongly the X-rays are diffracted by the molecule in different directions and from this you have to deduce the arrangement of the molecules. You make a measurement on diffraction and the result is the positions of the atoms.

Well, may I outline for you the nature of the problem in its full fearfulness. The molecule contains 10,000 atoms. Now, we have to find their positions

and their positions are defined by their coordinates, what we call parameters. So as we have defined X, Y, Z, the coordinates of each atom, there are 30,000 variables in our equations.

30,000 numbers which have to be determined. Actually it is not as bad as that because fortunately, if I may draw on the board for a moment, the molecule of hemoglobin has an axis of symmetry.

So if you know the position of an atom there, you know the position of its partner over there. So we have 15,000 variables only to determine. Now, we have enough equations to determine those variables

because each spot of the diffraction path gives us one equation. A function for the first spot of 15,000 variables equals something we can measure,

the amplitude of the X-rays that made that spot. And then you pass to the next spot, F2 and A2, and there are 30,000 of these equations. So that then is the problem, to solve 30,000 equations

which are more than sufficient to determine 15,000 variables. When we proposed to do this, I went to Melendy, who was at that time the head of the Medical Research Council. And I asked him for money.

That is, of course, why one goes to people like Melendy. And I said, the chance of our getting out this problem is practically zero. On the other hand, the importance of getting it out if we are successful is practically infinity.

And if you multiply zero by infinity, it is possible that you will get something. Please give me a great deal of money. And I'm very glad to say that he agreed, and that started our research.

Now, what was my part in this research? Because I think it is very wrong for heads of laboratories to talk too much about the work which their young people do. It should be left to the young people to do that. But I can perhaps again on the board illustrate what my part was, was WLB.

Going into a protein orbit was PIRS.

I think that perhaps describes it, that I was the first stage of the rocket which got this research off the ground. But this is the nature of a protein molecule.

It is very interesting the way in which nature builds up these very complex molecules which have to play a very specific part in our bodies.

Because the main characteristic of a protein molecule is that it does one task in the body and absolutely nothing else. One particular little bit of a chemical process

in our living bodies has its appropriate protein molecule, which does that and does nothing else. Now, instead of building a special structure for each of these protein molecules, nature has adopted a simpler device.

It is a device very like that which we use for making the many letters. Making letters serve for so many words. We have some 25 or 26 letters in the alphabet.

And with those, we can build words of any kind, conveying the most complicated ideas. That is not the only way in which you can do this. The Chinese, as you know, have a symbol for each fundamental idea,

a symbol instead of writing letters one after another, they draw a symbol which represents a word. Nature is European and not Chinese. She builds up these protein molecules with 20 simple amino acids,

small bits of chemical structure, not at all complicated. Rather conveniently, 20 can represent the letters of an alphabet. And the different proteins, just like again words with letters, are built by using these 20 simple building blocks

and arranging them end to end. Could I have my first slide, please? This slide shows the... What the lights are, I see. This slide shows these chemical bits.

They are slightly different on the right-hand side. That is what gives them their character. And again, like the letters which a printer uses, the little box which he puts to make a word, they are all the same on the left-hand side. It is these left-hand sides which can fit together.

They can... NH3 condense with a COOH of that one to form a bond by elimination of water, and then you can string these all in a row. And that is how the proteins are built up.

Each protein has a characteristic order of these little building blocks which then form a structure which we will be examining. My next slide shows the kind of picture given by the protein molecule. That is only a sample. It isn't quite focused.

That is only a sample. There are many other sheets of spots like that, but those are the spots we have to explain. One has altogether some 30,000 of those spots in the diffraction picture given by protein,

and those form the 30,000 equations. The positions of the atoms must explain the strength, strong or weak, of those spots. That was our material. And there are so many spots, it's clear there are more equations than variables, and therefore it should be possible to solve them.

And we looked at those for 25 years, trying to find out how to do it. Now, the method of attack. In early X-ray analysis, what one did was to move the atoms about in a likely way, to try various structures,

calculate how they would diffract the X-rays, and then match that with what was observed. Clearly it is quite impossible to try all positions of 10,000 atoms.

So that method was completely ruled out, particularly too because we had no idea of how they ought to be arranged. Nobody knew what a protein structure should be like. So we had to try another method, the Fourier method, which is now used for all complex X-ray analysis.

Could I have the next slide, please? That slide represents various musical notes. The variation of the pressure of the air in a musical note. And I use that just to point out

the principle of a Fourier series. The top one, which has had its head cut off, is the noise made by a flute. I think, ah, it is very simple. It's a very pure tone, just the fundamental note and one overtone,

that and that. This is a little more complicated. The next one's a clarinet. The next one is an oboe. And this horrible one here is a saxophone. Now, the Fourier series is just a representation

of the fundamental and the overtones, which you add together to get a curve like that. One can add together the fundamental and the overtones to produce the curve. Only the fundamental and the first overtone for the flute,

much higher overtones, say, for the oboe. Right, lights up, please. Now, a crystal. You can think of a not known, ah, lights. Least. Least. Thank you. Ah, now a crystal.

You can think of a crystal as a musical note in three dimensions, if you like. In just the same way that you can build a one-dimensional curve like that by adding fundamental and overtones. So in a crystal, you have periodic variations of density

in all directions in three dimensions. That way, and that way, and this way, and that way. Add those all together, and you get the density in a crystal. Because a crystal is a pattern and repeats, just like a note repeats.

When we examine a crystal by X-rays, we are measuring these overtones. Here is the unit cell of our crystal. We can represent that crystal like a musical note by waves running this way and waves,

another set, shall we say, running this way. And perhaps you can see that if the strata, if the wave has a big amplitude,

that this particular white one, then when X-rays are depicted from those planes, it will be strong. That's to say, a strong spot means a strong stratification, or Fourier element, of the crystal in that particular direction.

And if this one is weak, then when X-rays are affected from there, we would only get a weak reflection. In other words, the strength of each of those spots, which you saw in the picture, is a measure of the strength of the Fourier element,

which represents that repeat in the crystal structure. So we measure all these spots and can put together these Fourier elements, then we have the answer to our crystal structure. That sounds very easy. If that is all X-ray people have to do,

it is hard to see how they earn their salaries. But actually, there is a difficulty. You can, with X-rays, measure the amplitude of that component, but you cannot tell how much that way or that way it is in the crystal structure,

because wherever it exists, it gives the same X-ray reflection. To put it mathematically, you can measure these amplitudes, but you cannot measure the phases, not directly, anyhow. The strength of the spots does not tell you the phase. So X-ray analysis is a hunt for phases.

If we can only find out those phases, we can find out our structure. Well, now, for the next five minutes, I am going to tell you how proteins were not solved,

how we started up a blind alley, because it is a rather interesting, I think perhaps a certain interest in this story. When we started, we thought that the protein molecule probably had some kind of regular structure,

that the protein chains in it would be like, say, a series, a rows of these amino acids, regularly arranged. Why did we think that? I think because it was too horrible

to think that they were not, because it made it all seem terribly difficult. So we started off with this idea. Now, there is, I will not, I always think it is very bad, indeed, in a lecture to mention mathematics. One ought never to do that.

But just to give you, to sum up the results of a certain mathematical treatment, may I just tell you about something which X-ray analysts use, which they call a Patterson, after the name of the man who first pointed out the relationship.

Excuse me drawing so much on the board, but I always think better if I can draw at the same time. The real crystal, as I showed, is built up by adding Fourier elements. On the other hand, if you add up just these straight intensities of the spots

in a Fourier series, do not bother about things. You get a diagram which is called a vector diagram, or a correlation diagram. If in the real crystal, the actual crystal, there's an atom A there,

an atom B there, then in the Patterson, this vector diagram, starting from an origin here, that vector appears again down here of strength AB. The strength of A multiplied by the strength of B,

the mass of A, if you like, the mass of the atom at A and the mass of the atom at B, multiplied together, appear at that vector distance from the origin. Now, we made a Patterson of hemoglobin. The roots faithfully measured all the intensities and summed them up in a Fourier series of intensities

and got this diagram. Now this is the difficulty. There are, in the real crystal, ten thousand atoms. So in the Patterson, there are a hundred million vectors, because every pair of atoms gives you another vector.

So this is a picture of a hundred million vectors laid on top of each other, and at first sight it would seem you would not see very much in such a number. But, if there were a regularity, if in the protein molecule there were rows of these amino acids

parallel to each other, then we thought there'll be certain vectors which repeat so often that they will be over-mastering and we will see them in the Patterson in spite of there being this very large number. So we tried that. And the slide, next slide please,

shows what the Patterson looked like. And we thought, yes, there is something. There is the origin, and you can see stripes of density going along this way which might represent rows of these amino acids in the crystal. And we were very excited.

We were even more excited when Pauling, slightly later, proposed his alpha helix, which had the same repeat at about ten angstroms that the vectors seemed to show in this picture here.

My next slide shows Pauling's alpha helix. He said that when the amino acids join in a chain, the form of least energy is a corkscrew, or helix, going round and round. These are the different ends

which characterize the amino acids which you saw in my first picture. The common C-O-C-H-N-H of the chain is the backbone here which runs down and is coiled in a helical form. So we said, now this is wonderful, probably a protein is a set of Pauling helices

which are all parallel to each other like logs of wood in a bundle. And so there is a chance that we can find out how these lie and solve our protein structure. We were in a state of great excitement and we were quite wrong, because protein has no such simple structure at all.

There is a saying in English, perhaps in other languages too, that fools rush in where angels fear to tread. It was very fortunate that Perutz and I were not angels, because I think at this stage,

if we'd known how much harder the real thing was, how complicated, we would have stopped the research altogether. Now for the next stage, which had a partial success. I hope I can explain it. The crystal of hemoglobin has a series of shrinkage stages.

If you put it in mother liquor of different pH, it will shrink or expand while remaining crystalline, a rather marvelous phenomenon. My next slide shows the nature of this shrinking or expansion.

Two sides of the unit cell, the side this way and the side this way remain constant, but the third one, the angle changes and you get a series of stages of hemoglobin, there are more than this really, with different unit cells. Now perhaps without my going in it too far,

I can explain what my next slide means. Perutz measured the diffraction of all these shrinkage stages and plotted the results on one diagram like this.

Now you see our problem if we looked just at the diffraction picture of the hemoglobin in this projection. It had a hundred and fifty well-marked spots. Now these spots, each of these spots, represented one of these waves of density.

And as we were looking at the symmetrical projection, the one where there was an axis of symmetry, the corresponding waves of density have a phase either plus or minus. By symmetry, either the crest of the wave goes through the axis of symmetry,

or a trough of a wave. You can't have a wave in any position if you are to have a symmetry axis. So we had to assign signs, plus or minus, to about a hundred and fifty spots. And if you work out the number of waves of giving plus or minus to a hundred and fifty spots,

it is two to the power of one hundred and fifty, which was a very large number of possibilities to sort out. On the other hand, when we plotted this diagram, this is a point I hope I can make clear, when we had plotted this diagram,

it was clear that the diffraction by the hemoglobin changed, of course, as the form of the crystal changed. The spot appeared in a different place because the crystal axes had different angles. Now it is a very fortunate fact that if a quantity is plus or minus,

and if it changes through a zero value, it must be going from plus to minus. I hope that is mathematically correct. Mathematicians always have a way of getting round these things. But to a simple-minded physicist, it seems clear

that if a quantity is real, and can only be plus or minus, and if you find that it varies steadily and goes through a zero value, clearly it must be going from plus to minus, or minus to plus. So you see, although considering a single form of the crystal,

of a hundred and fifty spots, had two to the power of a hundred and fifty possibilities of signs, if we could draw this and say, now, if that's plus there, it's minus there, and it's plus there, that means that only we knew the sign of one of these loops, as we call them here,

the other loops in this row would be known. In other words, we had reduced the number of possibilities from two to the power of a hundred and fifty to two to the power of seven. We had reduced it by a factor of two to the power of one hundred and forty-three. A big reduction. But still, the number of possibilities remained rather large,

and we were stuck. Now, could I have the lights up please, Licht? I want to draw again, I'm afraid. This difficulty was got round by a brilliant discovery by Perutz.

And this is where Perutz left the first stage of the rocket and went off into orbit by himself. There is our protean molecule with its axis of symmetry. That protean molecule gives those diffraction results you saw there, in the last slide.

And we want to know the signs of those results. Perutz found that he could attach two molecules of mercury. Atoms of mercury. If you attach one, you attach two because the crystal has that symmetry.

Now, if you attach two molecules of mercury they add their diffraction to the diffraction by the protean. And the kind of pattern you get from two scattering objects are, of course, Young's fringes.

A series of fringes at right angles to the line joining the mercury atoms. Not there, but in the diffraction picture. I just draw them there to show they are at right angles to the line joining the mercury. So, Perutz discovered

not only that he could attach the mercury atoms to the protean molecule but that they made a difference in the diffraction a measurable difference he could observe. Could I have my next slide, please? This is a composite slide.

It is the diffraction by the protean without the mercury compared with the diffraction of the protein with the mercury. And the two slides have been displaced slightly so that the corresponding spots are just under each other.

Now, Ken, I hope you can see that in many cases the attachment of the mercury has made a difference in the intensity of the spots. Where is a good one to observe? There's a very striking case there where the pure protein has a strong spot

and the mercury one has nothing whereas when you attach the mercury it becomes strong and the protein compared to the protein, there's nothing. If you look at these pairs you see in many cases changes taking place in the intensity. Perutz was able to measure those accurately. Part of the success of this whole project

was that Perutz was a brilliant experimenter and could get reliable measurements. Now, if I could have my next slide, please you see that at once that tells us all the signs. Here are the mercury fringes sloping the mercury gives fringes that slope down this way

actually this slide, I'm afraid, is the wrong way round. Does it matter? Thank you. Now you see if that oh, it's upside down now never mind

if there is a plus fringe and it makes this get less that must be a negative loop on the diffraction pattern if on the other hand the plus fringe makes something go up as it does there that must be a plus fringe U, upside down there means it goes up D means it goes down

so you see by noticing whether the spot became stronger or weaker one could tell whether a loop was negative or positive here is another case a minus a trough of the Young's fringe making this go down

become less therefore that must have been a plus fringe whereas it made that one go up so that must be a minus fringe and so on, minus loop so we got all the signs and we put those signs into a Fourier series this case only a two dimensional one to get a projection

and for the first time we got a picture of the hemoglobin molecule the next slide shows this picture and it tells us absolutely nothing at all the trouble is that the protein molecule is so thick that it's about 30 or 40 atoms thick they're all on top of each other

and you really can't see anything but it was encouraging that anyhow if you did look at a row of protein molecules that is what they would look like it was something in the way of a success it was clear however that if we were to go on and get a real picture

of a protein molecule we must do it in three dimensions now that was hard could I have the light please but in principle it is possible again if I may draw a picture the unit cell

one of the Fourier components perhaps is like that but how do we know where it is it may be anywhere this way because that is the thing you cannot measure with x-rays but now you see if you put into the unit cell a heavy atom

mercury or gold or something of that kind iodine which you can do if you find an atom put in at A makes the spot stronger an atom put in at B makes the spot weaker because it's in the trough you see of these waves an atom put in at C

half way between the crest and the trough makes very little difference then you know you're right in putting the waves there of course I've turned it the other way round what you find out is A makes it stronger B makes it weaker C makes no difference therefore my waves must lie like this

to do it really properly you draw a vector diagram and make it all fit but that gives the principle so you see if you can find out the change in intensity when heavy atoms are put in like this you can find the phase but you need at least three of these heavy atoms to be sure of your phase

one is not good enough one atom was good enough for pyrupsis hemoglobin where we had worked out all these loops but when you have the general problem when the phase may have any value you need at least three heavy atoms to get a reasonable

equation to find the phase pyrupsis could not do that for hemoglobin it proved impossible at first and that is where Kindrew came in pyrupsis had the great idea of a heavy atom but it was Kindrew who went into orbit first and found out the structure of a protein because he found he could get

four heavy units stuck onto his protein one called myoglobin with that he worked out all the phases using of course a computer the computer had the task it is quite a task it had to form a series a Fourier series with 20,000 terms

and it had to sum up this series at 250,000 places inside the cell in order to get the distribution of density inside the cell then there was the problem when you had got all these densities as figures by the computer what did they all mean

my next slide shows how no, we finished with that next one ah yes that's right my next slide shows could we focus it right nice down this is a slide of Kindrew

trying to think what his results meant he took a large room in the laboratory he bought five kilometers of brass wire which he stood up on blocks of wood and then he got six young ladies who put little colored clips, blue meant very dense and red meant very little you see

on the wires cause from the results from the computer you could see what the density was so he assembled together all these little colored clips and then the wires were on blocks of wood that could be moved a little so that he could walk inside and he tried to see what that meant in terms of atoms

and you can see here he is beginning to build up the structure of the protein these wires represent lines between the atoms wherever the wires join there is an atom and he is piecing it together and finding little bits my next slide shows Kindrew contemplating the result

of all this work that is the structure of the myoglobin molecule that is a simpler molecule it's only got 2500 atoms in it this white thing represents the course it has a single polypeptide chain a chain of amino acids and this is merely

like the white line down the middle of the road which tells you where the road runs it just shows the direction of the chain the next slide is a better one to show the molecule if one wants to have a very good picture of something the thing to do is to persuade

the scientific American to accept an article cause it always draws most it always draws most beautiful pictures this is this is the myoglobin molecule there is quite a lot of poly helix in it you can see a bit like that and a bit like that this bit here

with its iron atom and what is called the heme group around it that is the place which holds the oxygen in myoglobin there is one heme group it holds one molecule of oxygen in hemoglobin and in hemoglobin there are four holding four molecules of oxygen the myoglobin has the oxygen in our muscles to keep

so that then is the success of Kendrew's results the first protein to be worked out well now I'll say a little bit more about the structure of another protein lysozyme this I'm interested in cause this was number two protein to be worked out

and was done in the laboratory of the royal institution by Dr. Phillips and his colleagues lysozyme is a protein which is an enzyme it has a very specific chemical job to do lysozyme is a protective enzyme in our bodies

it can attack certain kinds of bacteria and kill them and it attacks these bacteria and kills them by destroying their cell walls their walls of these bacteria have ribs in them very like cellulose

a compound very akin to cellulose and lysozyme is able to, as it were, bite these ribs in two to cut them it applies itself to the wall it catalyzes a change which breaks the cellulose chain by introducing water

and turning the usual chemical link COC into COH and CH so you COOH and CH and so you get a breaking of the chain lysozyme, we have lysozyme in our bodies as a protective enzyme it was discovered by

Alexander Fleming who at first thought he had discovered something with the properties of penicillin actually lysozyme is so present already in our bodies that we don't need any more and is not a medical help there is for instance a good deal of lysozyme

in our tears in the fluid of the eye I suppose to protect the eye Phillips has lent me this next slide which shows the first way in which lysozyme had to be produced fortunately it was discovered that egg white has a lot of lysozyme

in it and so this process was no longer necessary my next slide shows the nature of the lysozyme molecule it's a big molecule again nearly as big as myoglobin and it has a curious

cleft in it, I think perhaps you can see that running down there is a kind of valley, a valley in the molecule and opposite this valley are two very either sides are two very active units, glutamic acid and aspartic acid as amino acids and it is these

which are going to do the job of breaking down the wall of the bacteria my next slide again the scientific American has made the rest of the molecule rather faint so that you can see clearly the bit of the wall of the bacterium which has placed itself in this cleft

and that is what the lysozyme is going to break down now it is quite fascinating as Phillips has shown in the first place there are along the chain which is part of the wall of the bacterium there are units that can form hydrogen bonds

each of these units comes exactly opposite a molecular item in the lysozyme which can form a hydrogen bond so when the chain fits into this crack, everywhere there are hydrogen bonds to hold it exactly in place and to bring the weak point

of the chain which is going to be broken which is at this bend here exactly opposite the two active units, the two acids which are going to hydrolyze the chain and break it there so although we have now found out only a few of these proteins even these first examples show the nature of

a protein structure it is like a kind of machine tool such as is used in industry in the case of the lysozyme the hydrogen bonds are grips which hold the work in place in exactly the right place and bring the right point of the work

opposite the cutting instrument which is going to perform the necessary action these amino acids perform two functions in the molecule for the most part these amino acids are, how shall I put it something more than mere patting but they are space filling elements they are elements which

are just right to bring the active part of the molecule into the right conformation then the active part is exactly the right conformation to do the job which nature wants it to do Lewis has been examining the different forms of hemoglobin hemoglobin, there are many variations of the structure of hemoglobin

changes in the amino acids but he finds that these the innocuous changes the ones that don't matter different species of animals have slightly different amino acid contents of their hemoglobin the ones that don't matter are all patting, as long as

they have about the right shape they are all right but the ones which hold the chains together in hemoglobin and the ones which surround the place where the oxygen comes they are vital if one of those is changed the person is either very sick or dies some form of anemia

let's say there are certain vital amino acids which must be there to do the job the others which hold those in the right place you can change quite a lot and get away with it so as it were the picture of a protein is beginning to form as one of nature's machine tools which just do

this one specific job in the body now if I could have my last slide please have the next one that is the structure of hemoglobin that is the first crystal to be worked out to the same scale the structure of rock salt and when I look at this picture

I always feel how very fortunate I was to get the Nobel prize 53 years ago when the standard was so very much lower