AstraZeneca and LMB in Cambridge use one of world's most advanced microscopes to make breakthrough
Using a pioneering technique with one of the world's most advanced microscopes, scientists have made a ground-breaking biological discovery that could help develop new cancer treatments.
For a century, scientists have been using crystallography to examine molecules.
Now there’s a new kid on the biological block: A technique that’s being pioneered in Cambridge using a cryo-electron microscope – an incredible machine for which you “wouldn’t get change out of £5million.”
The Titan Krios is three metres tall, weighs about a tonne and operates at 200 degrees Celsius below zero.
There aren’t very many cryo-electron microscopes in the world. But Cambridge has three.
They have enabled researchers to uncover a fundamental secret about how DNA is repaired that could prove invaluable in developing new cancer drugs.
The study is a tangible example of how the collaborative approach to science being increasingly adopted in Cambridge is bearing fruit – and will make a telling difference to healthcare.
A consortium has brought together pharmaceutical companies, the University of Cambridge, the Medical Research Council’s Laboratory of Molecular Biology (LMB) and microscope manufacturer FEI.
Chris Phillips, associate director of structural biology at the IMED Biotech Unit at AstraZeneca, one of the pharma partners, told the Cambridge Independent: “We have all invested one way or another in hosting this microscope. That’s one of the nice things about Cambridge. This model is unique in the world – Cambridge offers an environment where these things can happen and they are not happening elsewhere.”
He believes the technique of cryo-electron microscopy, or cryo-EM, is set to become increasingly important in his field.
“Crystallography is fabulous and has been going for about 100 years but it has a major limitation and that is that you have to be able to grow crystals of the molecules that you’re interested in – and not all molecules can be crystallised,” he says.
Very large molecules and complexes of molecules fall into this category, while many proteins important in disease reside in a non-aqueous environment in cell membranes, making them particularly difficult to crystallise.
“So what the technique of cryo-electron microscopy does is it allows us to study all these sorts of molecules that we’ve really struggled to with crystallography before,” explains Chris.
“The specific molecule that we’ve studied here, ATM, is a classic example of that because it’s huge – 10 times as big as the typical protein you study by crystallography – and being able to produce it in sufficient amounts to grow crystals has proved impossible. But you only need a small amount of the protein for cryo-electron microscopy.”
Cryo-EM works by firing electrons through a very thin sample frozen in a liquid ethane. The machine collects the 2D images from which a 3D shape is calculated. It boasts near-atomic resolution which, in this study, means four-tenths of a millionth of a millimetre.
“You have to flash freeze the molecules in a very precise way,” explains Chris. “They have to be in a single layer of vitreous – non-crystalline – ice, an ice glass, which is no thicker than the molecule itself.
“The signal you get from the electrons hitting the molecules is very, very low so you need this really thin film to be able to detect it.”
The research on ATM – human ataxia telangiectasia mutated, to give it its full name – gave Chris the chance to use a cryo-electron microscope for the first time. Two of Cambridge’s are housed at the LMB and one is at the Nanoscience Centre on the university’s West Cambridge site. But what is it like to use one?
“It’s all encapsulated,” he says. “When you open it up it’s a huge array of wires and cables, and a big vacuum tube. It’s very complex. But once you’ve loaded the samples in, you interact with it through computer software.
“The LMB are world leaders in this and some of the scientists there have pioneered this technology.
“They’ve had the equipment there for a few years and have done very well.
“It’s been fabulous for us as a company to be able to collaborate with them. It’s enabled someone like me to get into a new technology, to define a strategy for how a pharmaceutical company can access it, use it and how it can impact our drug discovery programmes and we’ve got this great study out together.”
In a paper published in Science Advances, the scientists explain how they used cryo-EM to define the structure of ATM in different states.
Chris explains: “The study is all about the mechanism by which DNA repairs. The genome can be quite unstable, particularly in cancer cells. You get DNA breaks.
“Think of the classic DNA double helix structure. When both strands are broken and your genome is fragmented, that’s a highly toxic situation and the cell has to be able to repair that break very quickly. That’s what the ATM molecule does – it triggers the repair of the double strand breaks.
“In the cancer setting, if you can inhibit that process and stop that repair, you can kill the cancer cells. You are preventing the cancer cell repairing itself.
“What we’ve done with the LMB is really about the fundamental biology behind this. It’s understanding the mechanism by which the process is triggered.”
Algorithms are used on the images created to reconstruct the molecular structure of the protein, opening up the possibility of creating drugs to target this repair process.
“Another great difference between cryo-EM and crystallography is that in the latter you get one structure for your molecule – one shape. With cryo-EM, you’ve frozen the molecule. Any changes it makes in shape you can record so you can get more than one structure of the molecule. You can get a dynamic view of the different shapes the molecule can make,” says Chris.
For this first time, the incredibly detailed images revealed that ATM acts as a molecular switch.
“What we’ve seen in this study is two different shapes that the molecule can make: One in which it will not be active and one in which it is active,” says Chris. “So we’ve found a way in which this molecule is switched on. Understanding how biological processes are controlled by these mechanisms allows us to think of ways we can take advantage of that in drug discovery. We could target it in both states to make a drug – we could target the off state and stabilise that so that would stop the molecule triggering DNA repair. Or you could target the on state and disrupt it.”
This is a field in which AstraZeneca, which will move into its new HQ and R&D centre on the Cambridge Biomedical Campus next year, has great expertise already.
“AstraZeneca has a big investment in DNA damage response and we have a number of interesting molecules in early clinical discovery. That’s why this collaboration with the LMB has gone so quickly because we have great reagents from our drug discovery programmes and they have great technology and a lot of biological understanding in this area. The two have come together and it’s been very quick to get this study out,” says Chris.
“It’s the first time we have a real understanding of how this mechanism works. There’s a real pure science step-change for this area of clinical study.
“To do something for the first time is really hard work but it’s fabulous. Working with scientists at the LMB for somebody within the industry is really exciting. I’ve been an academic in my time as well so to do this sort of work has been great for me. It’s why we turn up to work – to discover new stuff. It’s a huge drive.”
Chris is confident that cryo-EM will play a key role in future research.
“There’s a debate in the field about this but I think it’s pretty clear now that cryo-EM is going to be a core technique within structural biology and it’s going to have a huge impact on our understanding of molecular processes.
“It’s happening very quickly. If you’d asked me a year ago, I wouldn’t have been as confident about that. Now I’m very confident,” says Chris, who is based on the Science Park.
So what’s next?
“We work in highly multiple disciplinary teams with chemists and bioscientists, so what we’ll do with this information is build models on how drugs can interact with this protein and make new molecules on the basis of that.
“It’s an iterative drug discovery process.”
Postdoctoral scientist Dr Domagoj Bareti, who performed the analyses, says: “With this work, we have provided a new framework for understanding the ATM biology.”
Roger Williams, of the LMB, adds: “The collaboration with AstraZeneca has been an amazing experience. At every step of the process we learned from each other and shaped the approaches to our common goal.
“The project got a fast start because we were able to take advantage of the expertise and reagents that AstraZeneca had developed for ATM.
“Using the world-leading cryo-EM facilities in the LMB and our expertise with structural biology of related enzymes, we swiftly revealed the structure of this key enzyme in the DNA damage response.”
A world first that brings pharma companies and researchers together
The Cambridge Pharmaceutical Cryo-EM Consortium is the first of its kind in the world.
Launched in April 2016, it brings together five pharmaceutical companies – Astex Pharmaceuticals, AstraZeneca, GlaxoSmithKline, Heptares Therapeutics and UCB – with the Medical Research Council’s Laboratory for Molecular Biology and the University of Cambridge Nanoscience Centre, along with microscope manufacturer FEI.
FEI’s Titan Krios cryo-electron microscope has been installed at the Nanoscience Centre on the West Cambridge site.
Mike Snowden, VP and head of discovery sciences at AstraZeneca’s IMED Biotech Unit, says: “Having access to this technology through the combination of our MRC collaboration and our unique pharma consortium really helps keep AstraZeneca at the leading edge in the industry.
“Cryo-EM still has some way to go before it becomes routine, but to be part of an organisation investing early, collaborating with the best external groups in the world, and positioning it for the first time within the drug discovery setting is really exciting.”
More by this authorPaul Brackley