A discussion around a Titan Krios cryo-electron microscope

Focusing on the molecules of life with cryo-electron microscopy

The biochemical processes of life are being revealed in atomic detail using cryoEM, a sophisticated new microscopy technique which is revolutionising the field of structural biology.

Scientists at the Crick are using a technique called ‘cryo-electron microscopy’ or cryoEM to image complex biological processes in atomic detail. 

The multimillion-pound microscopes use focused electron beams rather than light, resolving greater detail. Using electrons, they can see down to the molecular systems at work in our cells and tissues. Flash-freezing samples to around -190°C using liquid nitrogen preserves biological structure and gives the technique its name: ‘cryo’ means cold or frozen. 

If you know the structure, you know how to break it. You can design a precise drug or molecule to impede the process.
Peter Cherepanov, Group Leader

CryoEM has improved significantly in the past few years, moving from seeing proteins as fuzzy blobs to resolving the full molecular structure. These advances were recognised in the award of last year’s Nobel Prize for Chemistry to the three scientists who developed the technique. 

The Crick has a set of these microscopes available for research in its Structural Biology science technology platform, led by Phil Walker. 
 

Helicase molecular structure

Molecular structure of a helicase as it begins to unwind DNA. Image by Ferdos Abid Ali in Alessandro Costa's lab.

CryoEM at the Crick 

The Crick’s cryoEM facility is located in the basement, 20 metres below ground. The 4m-high box enclosing the microscope manages the temperature, air flow and vibrations that could disturb the images collected. 

The whole microscope is set on a base mounted on compressed air that actively dampens vibrations and disturbances, while metal in the walls of the room shields the microscope from stray magnetic fields. Automated computer control of the microscope allows data to be continuously collected overnight and to be operated remotely. 

The Crick has two of these state-of-the art Titan Krios instruments, funded by the Medical Research Council, along with several smaller instruments to support a wide range of science at the Crick. 

Thanks to related Wellcome funding, a third Titan Krios microscope will also be housed at the Crick for use by a consortium of London universities and institutes: Imperial College London, the Institute of Cancer Research, King’s College London and Queen Mary University of London

This collaboration should see further sharing of skills and expertise in cryoEM among the research community in London. The large investment in these microscopes by the Crick, its founders and collaborating organisations reflects the depth of insight cryoEM can now give into biological processes. 
 

Resolution revolution 

The improvement in the microscopes has come from a range of developments. New cameras that are much more sensitive, better sample preparation and new data processing approaches have all come together to give a significantly greater level of detail with cryoEM.

The Titan Krios instruments are managed by Andrea Nans, who has been working with cryoEM since 2005 when she did her PhD in the USA. 

CryoEM has allowed us to look at very large and very complex molecular machines in ways that would be really challenging if we were to use protein crystallography. 
Alessandro Costa, Group Leader

She remembers the rigmarole of collecting data manually on photographic films before the new cameras came along. “It was very painful,” she says. “It’s easier now, especially as more people want to get into cryoEM.” More people are interested because of the unexpectedly rapid advances in what is now possible with the microscopes. 

“There’s been a revolution in imaging biomolecules and more surprises are ahead,” says Peter Rosenthal, a group leader and coordinator overseeing development of cryoEM research at the Crick. 
 

Form and function

It is the jump in resolution that is changing what is possible for scientists. They can now use cryoEM to determine the position of atoms within large biological molecules. 

The field of structural biology involves determining the three dimensional shape of biological molecules that carry out key processes in cells. Understanding how the biomolecules look is important because it gives information on how they function.

Peter Cherepanov, Group Leader at the Crick, explains: “Everything in biology is based on chemical reactions and the interaction of molecules. If we can determine the atomic structures, very often it explains the mechanism through what contacts what.” 

Integrase molecular machinery

Structure of the integrase molecular machinery from a virus related to HIV. Image by Peter Cherepanov. 

He says this offers important insight: “If you know the structure, you know how to break it. You can design a precise drug or molecule to impede the process.” 

For decades the method of choice to determine structures was X-ray crystallography. For this, you need to purify large amounts of your protein molecule of interest and find conditions in which it crystallises. You can then use X-rays to work out the protein’s atomic structure when lined up in an ordered crystal. 

Crystallography has been tremendously successful in telling us how proteins carry out their role, in explaining the effect of certain mutations and in drug design. 

But suddenly, in just the past three or four years, cryoEM is providing the same kind of detail without the need for crystals. And it can do so with large assemblies of proteins as they are when active in the cell – something unobtainable before. 

“Our job has suddenly become more exciting,” says Alessandro Costa, another of the structural biology group leaders at the Crick.

“CryoEM has allowed us to look at very large and very complex molecular machines in ways that would be really challenging if we were to use protein crystallography.”

Stories

Exceeding expectations

Peter Cherepanov

Peter Cherepanov has seen what is now possible with cryoEM. His group works on HIV and other members of the same virus family. 

They have been particularly interested in one viral protein called integrase which carries out an important stage in the virus’ life cycle. It catalyses the insertion of the HIV genome into the DNA of our own cells where it can hide and stay dormant before re-emerging at a later time. A drug that stops this integrase step would be a useful addition in tackling HIV/AIDS. 

The group had been using X-ray crystallography to study the structure of an integrase from a virus related to HIV. Now they have moved to use cryoEM, and they still get the same level of fine structural detail. They can see how the integrase works and see potential drug molecules bound inside. 

“This exceeded my expectations,” says Peter. “I knew it was possible to see small molecules in the cryoEM. But this level of detail? People have been impressed: not everyone realises it is possible like this.” 
 

Watching biochemistry happen

Alessandro Costa

Alessandro Costa leads another structural biology group at the Crick. His team studies DNA replication, working on the structure of a molecular machine called a helicase. This is a ring that encircles DNA and unwinds it for another machine called a polymerase to come in and duplicate the DNA. 

His group has been able to use cryoEM to capture the structure of a helicase bound to DNA in various states. From these images, it is possible to get insight into the mechanical steps involved as DNA is first unwound then replicated. 

But Alessandro wants to move from static images to film a biochemical movie. His lab is currently carrying out experiments to understand the time resolution they would need to achieve to see the various steps in the duplication of DNA. That will enable them to tailor an experiment for the cryoEM to see the different events and time points in the process.

“We now want to visualise DNA replication as it is occurring, to see DNA as it is newly made – all at close to atomic resolution. This really is visual biochemistry.”
 

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