Rosalind Franklin has always been a personal heroine of mine, both as a scientist and as a feminist. She was integral to the discovery of the double helix structure of DNA, and her work in X-Ray Crystallography was pioneering. So when I got an email from Vicky saying that on my work experience, I would be spending a morning working in Professor Brian Sutton‘s X-ray Crystallography labs in the same department that Franklin worked in, it was fair to say I was pretty excited!
However, although I have always admired Franklin’s work, I had only the shakiest understanding of what on earth X-ray crystallography even was. During my morning in the Randall department of Bio-physics, Katy and Sneha, two women working in the department, explained to me what they do in the labs, and how it all works. I’ve tried my best to explain it here. Spoiler alert – it’s even more confusing than the name suggests.
The Biology The best way to explain a protein is to say it is a lock and key. Each protein binds with a partner in a specific disease, so it needs a specific drug to fit into the lock and block the disease. In order to produce enough different proteins to study, and to develop new drugs, you have to grow them in bacteria. You replace a section of the bacterial genes with those of the protein, and the bacteria grow. Bacteria reproduce by splitting into identical copies of themselves so each new bacterium will contain the protein you want. In this way, we can express large quantities of proteins with relatively little effort.
The Chemistry However, they then need to be purified. This is done using chromatography. Huge channels are made, lined with nickel. The protein is bound with a histidine tag and fed down the channel. The histidine tag binds with nickel, binding the proteins to the sides and leaving all the impurities to be washed away. You then remove the nickel and the tag, and are left with pure protein.
You then need to crystallise the proteins. The protein is put into a round well next to a reservoir and mixed with a higher concentration of the substance that you want to bind with the protein than the concentration of the protein. Due to the process of equilibrium (of which I understand enough to pass Chemistry GCSE but not enough to explain) the protein becomes more concentrated, and eventually crystallised. However, this process is also down to a lot of luck. The protein crystals are then frozen in liquid nitrogen.
The Physics Electrons scatter waves, and in a microscope, lenses focus the scattered light rays, meaning you can see the image of the object you are focusing. X-rays are types of waves. However, they have a much higher frequency than light rays. They are also much stronger, meaning that there is currently no lens that can focus them, so we cannot use a microscope to see things diffracted using X-rays.
Crystals amplify the signal given out by the X-rays. The definition of a crystal is that it is an ordered arrangement of atoms, in this case proteins. This produces an ordered diffraction pattern. When waves pass through a crystal, they produce a diffraction pattern made up of different spots. Each spot corresponds to a point in the lattice, and represents the amplitude and the phase. Due to the phase problem (http://www-structmed.cimr.cam.ac.uk/Course/Basic_phasing/Phasing.html) it’s difficult to determine the structure of the protein, so lots of complicated maths goes on at this point, usually on a computer. The protein’s structure is then mapped.
Why Is It Important? X-ray crystallography maps proteins. Each pathogen has a specific antigen on the surface of its cells, and the immune system creates antibodies (which are proteins) to latch onto these antigens and destroy the pathogen. If we can map proteins, we can work out which ones lock onto the antigens, coming back to the idea of a lock and a key. This way, we can create drugs that successfully destroy pathogens in the same way that the immune system does, leading to more and more diseases being eradicated over time.
There. That wasn’t too complicated now, was it?