Biology on the Atomic Scale

Danny - My name is Danny Axford and I'm a support scientist on beamline I24 which is a microfocus macromolecular crystallography.

Meera - What kind of research is done here? What can be looked into here?

Danny - Primarily we're looking at the atomic structure of biological molecules. So these are molecules that are fundamental to how life processes work and I24 in particular has been designed to look at the most challenging samples that have so far proved difficult to analyse. So we use an especially highly-focused x-ray beam which allows us to probe smaller samples, typically we're analysing crystals and the more complicated the molecule the harder it is to produce a crystal and when you do get a crystal it is typically small. So we've designed our x-ray beam to be focused as small as possible which helps with this analysis.

The idea with macromolecular crystallography is in order to get a strong enough signal from the diffraction experiment, you grow a crystal and the crystal is an ordered lattice of the molecule and the bigger the crystal is, the stronger the signal you'll get. Now complicated molecules really don't like to grow into crystals. Typical examples are membrane proteins - these are typically embedded in the surroundings of cells so they're involved in transporting molecules in and out of cells and because they sit in the membrane they are not water soluble. So to grow a crystal from solution is very tricky.

Meera - Why does this feature make them harder to get into crystal format then?

Danny - Because typically they would be surrounded by the lipid that forms the membrane and we're not interested in the lipid, we just want to look at the protein, the protein itself. But, if you remove too much of the lipid, then these molecules then just fall apart. What you have to do is replace the lipid with detergent which allows you to solubilise these molecules and then grow them into crystals.

Meera - And they form quite small crystals?

Danny - Typically they will be very small, often very thin so you often get plates. So they are sort of 2 dimensional and this lack of volume means that the signal that you can detect from these crystals is very, very weak.

Meera - So why has seeing such small crystals been difficult with other beamlines and how has this beamline overcome those problems?

Danny - Typically other beamlines would have an x-ray beam of maybe 50 to 100 microns whereas on I24 we've managed to get our beam below 10 microns, we're now working towards 2 or 3 microns and if your sample is only 2 or 3 microns and you hit it with a beam that is maybe 100 microns then most of the x-ray beam will be missing the sample and that is essentially just adding 'noise' into the signal that you detect. So the signal that you are looking for is just washed away by the noise. Whereas if we can reduce the beam to the size of the crystal itself, then the signal to noise ratio is increased massively and so the very weak signal becomes visible when otherwise it wouldn't have been. So we can actually have a look at the beamline and see the components in action

Meera - So we've come through to the beamline now and there's a large metal box basically in front of which there's another attachment out of which the beam, the x-ray comes out and then in front of that there's a very small, miniscule looking pin which the sample is placed. So how small is this sample again?

Danny - Ok, so some of our samples are just a few microns in size so we're talking less than a tenth of the size of a human hair. So even under the optimal microscope that we're got integrated into the beamline, these samples are very difficult to make out.

Meera - This large metal box, this very thick metal box, behind it is where this beamline comes into its own I think, it is quite unique

Danny - That's right, inside this vessel here we've got an extra set of focusing mirrors. That allows beamline I24 to get the really small microfocus beam required to hit these samples.

Meera - So that hits on to the sample. The pin that the sample is on is just a couple of centre metres in front of where the beam comes out, but then that's diffracted onto a big, about half a metre squared, board about a metre and a half away?

Danny - That's right, this is a detector. We can actually move it closer in, it's extremely sensitive and it can read out very quickly.

Meera - You mentioned that an example of a complicated molecule is a membrane protein but what other examples can you give, what has been looked at so far on this beamline?

Danny - Ok, so another good case are virus particles in samples. These typically are very large molecules, in the order of millions of atoms. Typically they won't freeze very well at all so often instead of freezing the samples, we have to collect the data at room temperature and that means that the samples do not last for very long at all. They are destroyed by the x-rays inside of a second which is why we need the sensitive detector which is very fast and typically we'll have to get through lots of samples and then try and combine the information we get from them into one complete picture.

Meera - So viruses, membrane proteins, so largely biological molecules?

Danny - I mean in some cases we will shoot small molecules, which could be in the form of drugs for example, but these would be attached to the biological molecules, so we can see where the drugs attach, that allows us to maybe 'tweak' the design of the drugs to have a better interaction with the target molecule.

Meera - Danny Axford, Beamline Scientist at Diamond's I24 microfocus macromolecular crystallography beamline