Understanding Crystallography: Part One

From protein to crystal

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A guide to crystallography

How can you determine the structure of a complex molecule from a single crystal? 

Professor Elspeth Garman take us on a journey into the world of crystallography - from protein production and purification to growing the right type of crystals.

In her laboratory at the University of Oxford Elspeth introduces us to some of the tricks of the trade, key techinques and the machines that help her team grow crystals ready for X-ray analysis at specialist facilities like Diamond Light Source. 

This film was supported by the Science and Technologies Facilities Council (STFC).

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Materials

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Type:
Documentary
People:
Professor Elspeth Garman
Location:
Oxford, UK
Published:
2014
Filmed:
2013
Credits:

Ed Prosser ;/ Royal Institution

Collections with this video:
The Crystallography Collection

cc_by-nc-nd License: Creative Commons

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  • Model of Lysozyme at the Royal Institution.

    Image: Paul Wilkinson
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Transcript

This is a three dimensional structure of a protein called lysozyme. It represents the order of nature. And I feel awed when I look at it.

It's an enzyme that's in our tears and saliva and mucus, and it helps us fight bacteria. And knowing this three dimensional structure helps us to understand the mechanism of action of this enzyme, and therefore how it helps fight bacteria in our bodies.

Unfortunately, the size of the molecule is such that it's far too small for us to see under a light microscope, or with our naked eye, because the wavelength of light is much, much larger than the size of this tiny molecule.

For that reason, we have to use radiation that's much smaller in wavelength. And we use X-rays to look at these molecules.

These are protein crystals. Locked within each crystal are millions of protein molecules, all arranged in an ordered, grid-like structure.

By firing X-rays at these, and measuring how they scatter, we can work out the molecular structure of nearly any crystallised sample. It's through this method, known as X-ray crystallography, that some of the most important biological structures have been obtained. from the double helix of DNA to numerous proteins, vitamins, and drugs.

But getting from a crystal to something like this, the structure, is not at all trivial. And it can take a long time to grow a suitable crystal.

Now we're in the Protein Production and Purification lab, because before we can set up crystallisation trials, we need to produce enough protein for that process. We do this by genetically modifying E. coli bacteria, which then acts as a little factory to produce our protein in a large flasks of broth, which we incubate.

Once that's happened, we break open the cells, extract the protein, and then purify it, ready for the crystallisation process.

So why do we need to grow crystals of our protein molecules before we can shine X-rays at them and try and find the three dimensional structure of them?

Well, if we imagine that this string of beads is a protein molecule made up of 20 different amino acids-- different colour of beads-- that are found in nature, this folds up in a very complex, complicated manner in the three dimensional shape, like this.

And if we look at one of these-- a true biological molecule here, a real one-- what this metal model represents is a string through the beads. And you can see it starts at this end, and it follows a pretty torturous path going around here, that you'd never imagine when you just look at the string of amino acids. And the other end of it comes out here.

Now it turns out that if I take a tube here with my protein in it, that I've purified and prepared, there's millions and millions of protein molecules in there. And if I shine X-rays at it, the X-rays will scatter off in all sorts of random directions. And I won't get any information about the shape of the molecule within the tube.

However, if I can get the protein molecules to line up in an ordered array-- such as in a crystal, where they're all lined up in the same orientation-- when the X-rays scatter from the crystal, then I can get enough information. The signal is strong enough for me to get the three dimensional structure of the protein.

We've now come down to the crystallisation lab to look at how we crystallise proteins. In this Petri dish, I've got some supersaturated sodium acetate. And that means that there's so many molecules crowded in this solution, it's almost not holding the molecules. And it wants to solidify.

And if I hit it with a spatula here, you can see that it crystallises. We get a fantastic pattern as it crystallises across the dish.

Essentially, this is what we try to do with our proteins. Which is to produce a supersaturated solution of the protein, and we dehydrate in a very controlled manner.

The proteins we work on here unfortunately can be sometimes really difficult to crystallise. So we load small volumes of the protein into trays like this, with different additives. But we have robots that help us do that by pipetting small volumes into these trays.

Once we have the tray with the protein and additives in, we take it around to this crystal hotel which holds the tray for several weeks at four degrees centigrade. And also monitors whether we have crystals or not by photographing the tray drops regularly.

But that's only part one of the story because once we finally manage to grow a protein crystal, we then have to take it for X-ray analysis. And from the data we obtain, we try to generate a structure of our protein molecule, such as this one of lysozyme.

But the protein structures we work on today are far more complex. And they can produce very small and delicate crystals. So to study them, we have to take them to extremely powerful X-ray sources at specialist facilities, such as the Diamond Light Source. It's only once we get our crystals there that the next stage of our journey can truly begin.

It's the most expensive and sophisticated scientific facility ever built in the UK. The instrument in this building can produce X-ray beams powerful enough to peer right into the atomic heart of all kinds of matter.

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