At this stage... I suggested this and I did say this in the lab and told Tony Durham, that we ought to use the disc for the assembly. Now, Joe Butler had joined us by this time... Joe Butler, of course, because Reuben was leaving. He was leaving to... he left, I don't know, when did he join you in Germany?
[Q] '69.
'69, so this was '70, so he'd left by them. So he... and I got Joe Butler who was a biochemist, a protein chemist to join us. So I asked... I asked Tony Duhram, who was my research student to do some experiments and make some RNA, probably do it with Joe... Joe Butler and to mix the disc with the pure viral RNA and see if it nucleated assembly, and started the assembly. He wouldn't do it because he'd been told by another research student unit of mine called Peter Gilbert, who was a rather toughy and very determined, who'd been carrying out three-dimensional image reconstruction on discs. And came to the conclusion that the two layers of the disc related by a two-fold axis of symmetry perpendicular to the axis of the disc, which meant that the two layers were back to back. Now... so that meant that... only one layer would be active, but you see in the... if a disc is to turn into a helix then the... the two turns had to be facing the same way, if it doesn't have a two-fold axis for symmetry. Peter Gilbert, being a very persuasive fellow and being a fellow researcher with Tony Durham said that I was, that my ideas wouldn't work at all, that's why he wouldn't do the experiments, he wouldn't do it. So I actually ordered, I don't often have to do this, I ordered Tony Duhram to bring me a solution of acetic acid. And he brought the acetic acid and I poured it, it's one of the few things I ever did with my own hands. I poured the acetic acid into this... into the solution of discs, it all went cloudy instantly. And then we called you and you looked in the microscope and what you could see were large aggregates of dislocated discs on top of each other. And so it was fairly obvious that if protons could do it... turn in changing the structure of the, it was a very facile thing to turn the disc... turn the disc into helix, just like... just like that. And the... And so... but then, Joe Butler was around, so we got Joe to do the experiments, Tony carried on with doing other things as well. This showed clearly that this was the nucleating agent, so then... we... we published a paper on the... on the discs, you and I and Tony Durham about the discs dislocating and forming helices. And over some time they annealed into a perfect helical rod, and so then I got Joe to make some... RNA and asked him to... he'd just joined, to just see if the discs would nucleate the assembly of the protein. So we... we added... the idea was we would nucleate the assembly, so we added the... Joe did these experiments, he made a solution of discs, we knew how to make them in the pH7. He added the RNA and within five or six minutes it began to... it went cloudy. You could see it by eye, I mean, of course it had a light scattering so it was clearly working. So I assumed at the time that the... that the agent would be that the protein contains small aggregates, so to start nucleation and the aggregation or rather the elongation as a biochemist would call it or the growth as a physicist would call it would take place by adding one unit at a time. And this turned out not to be true at all because the... the more discs, when... when we changed the concentration of the discs it went faster and faster and made a more or less a complete virus. And so, the discs were being involved not just in nucleation but also in the growth in dislocating. And so the... And so that is something... so we were able to make more or less complete viral particles in about six minutes compared to the 24 hours. Now, the recipes for making, reconstituting the virus out of protein and RNA, if you looked up Frankel-Conrad's papers, they added some salt to the protein and they waited for several hours before adding the RNA. And I... of course it was obvious what this meant, they were adding the salt to form a disc.
