Cryo electron microscopy, or cryo-EM, has been around since the 1970s, but it’s only recently, thanks to technological advances, that the technique has become more accessible. The technique came to public attention in 2017, when the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank and Richard Henderson in recognition for their work in developing cryo-EM.
Cryo-EM in the Nordics is now becoming more prominent, thanks to the establishment of facilities in Stockholm, Umeå, Copenhagen, Aarhus and Helsinki, and the launch of initiatives such as the CryoNet network, which involves researchers from Nordic EMBL Partnership members, the Laboratory for Molecular Infection Medicine Sweden (MIMS) and Danish Research Institute of Translational Neuroscience (DANDRITE).
Even though low-resolution cryo electron microscopy has been used at the University of Oslo's Department for Biological Sciences since the early 1980s and recently at the Norwegian Radium Hospital, University of Oslo, Norway does not yet have a dedicated cryo-EM facility for high-resolution single particle analysis. We interviewed NCMM’s acting Director, Hartmut Luecke, and researcher Eva Cunha, about their plans to bring the technology Norway, and why it is so important for their research. We also include a case study from Nordic EMBL Partnership sister centre, DANDRITE, on how their cryo-EM facility came to be.
Can you tell me a bit more about your experience with cryo-EM, and how it all works?
Hartmut Luecke:
I came to cryo-EM relatively recently. I’m a structural biologist by training, and was primarily trained in a technique called x-ray crystallography. This has traditionally been our ‘bread and butter’ when it comes to determining the detailed atomic-level structures of proteins. However, in the past five years or so, another technique has emerged that has significant benefits when it comes to studying the structures of proteins, and that is cryo electron microscopy, or cryo-EM.
To be able to examine the structure of a protein, you need a pure sample. In other words, your target protein needs to be isolated from all the other things that are floating around in a cell. If you have a large amount of pure sample (i.e. milligrams), you can use x-ray crystallography, which means you need to crystalize the protein of choice in order to be able to image it. It can be hard to generate these crystals, and it’s not always guaranteed that you’ll be able to produce them. This is where cryo-EM comes in.
One of the main benefits of using cryo-EM is that you can get away with a much, much smaller sample, and you don’t need to crystallize it.
Your sample is placed on a cryo-EM grid - a tiny grid 3 mm across – which is then prepared using an instrument known as a vitrification robot, or ‘Vitrobot’. This automates the process by firstly blotting the sample to remove excess liquid, and then plunging it into liquid ethane (negative 180 °C), thereby snap-freezing it.
This vitrification process is also another advantage of cryo-EM – your sample is basically your target protein in solution frozen in time, so the contents are effectively as close in terms of appearance to their pre-frozen state as possible. The sample then needs to stay at the same super-low temperature, even when placed in the microscope, which can be quite a challenge. We’ve recently taken delivery of a Vitrobot, so we’ll soon have the capacity to prepare samples in this way here at NCMM.
Eva Cunha:
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I did my undergraduate degree in biochemistry in Portugal, and after that a biophysics PhD at Johns Hopkins in the USA. Then, in 2013, I decided to move into structural biology for my first postdoc at Biogune in Spain and as a result, started working with crystallography. Around this time cryo-EM really started to boom, mostly thanks to big improvements in image resolution, which meant a big jump in what we were able to see in terms of the structure of a protein. What was previously only available with crystallography, also became possible with cryo-EM.
I realised that cryo-EM was going to be huge for structural biology, and in 2015 I decided to move to the Max Planck Institute in Frankfurt to learn more.
This is where I did my second postdoc, and also where I learnt all the basics of how to operate cryo-EM microscopes. I was also able to learn more about what was possible using the instrumentation, and further realise its huge potential.
It was during my time in Frankfurt that I applied for a Marie Curie Individual Fellowship. The project that I successfully applied for involves solving the structure of a membrane protein with cryo-EM, so currently I have to travel to Sweden, Denmark or Germany to use their microscopes. They all have wonderful facilities, but I’m sure that we can create something equally strong here in Norway.
What are the plans to introduce cryo-EM to Norway, and what challenges do you think you will face?
Hartmut Luecke:
Norway does not currently have a dedicated cryo-EM facility, so the main challenge, as with setting up any type of new facility, is funding. In order to be able to install a cryo-EM, a wider national consortium, together with the EM facilities at the Department of Biological Sciences, UiO and the Norwegian Radium Hospital, UiO, NCMM and the universities of Bergen, Tromsø, and Trondheim have recently applied for an infrastructure grant from the Research Council.
If successful, we will install an Arctica cryo electron microscope here in Oslo in late 2019. The Arctica is somewhere in the middle between smaller, more basic cryo-EM microscopes and the most sophisticated one, called Titan Krios. The dream would of course be to have a Titan Krios microscope in the future, as they have at DANDRITE (Aarhus) and MIMS (Umeå). These produce a higher resolution, which also means they naturally require a larger budget and a specially adapted space.
The plans for the University of Oslo’s new Life Science Building, which is due to open in 2024, have already been altered to accommodate two Titan Krios microscopes, with provisions for electromagnetic and mechanical shielding, and the Arctica microscope applied for eventually will be moved to the new facility.
Furthermore, cryo-EM microscopes generate a huge amount of data which then requires processing, so we need a strong IT infrastructure in place. The IT team at NCMM has already laid the groundwork for this by installing the 250 Terabyte storage server and a GPU compute server, and we have been processing cryo-EM data we collected elsewhere. With this, and the Vitrobot, we’ll have the capacity to prepare samples and process data.
Another point is that finding the right researchers and engineers to operate such a facility can pose a bit of a challenge, as the field is still relatively new. However, thanks to the current momentum and attention given to cryo-EM, I’m sure this will soon improve.
How would having a facility here in Norway benefit your research?
Hartmut Luecke:
We have been approached by several people who would like to collaborate with our group, so I think having the proper cryo-EM instrumentation would really help drive this forward. I’d also be really excited to open up the facility for use by other researchers. We would like to train others in cryo-EM, and show them how to process their samples – much like a standard core facility.
I also think that bringing cryo-EM to Norway would help to drive more successful ERC grants applications, along with helping us to secure more high-profile publications. It would also benefit Norway’s next generation of scientists; to have a thriving cryo-EM community here would mean we could take the lead on helping to train some of the future stars of structural biology, and help to drive further innovation and collaboration in the field in the Nordics.
It would also be great to not have to travel to use facilities in other countries, as we are currently doing. As the samples need to remain frozen well below minus 120 °C, they are quite difficult to transport. This has meant taking them by road to places like Umeå, which takes a lot of time.
How can cryo-EM be used in ‘real world’ applications?
Eva Cunha:
One aspect that’s very relevant for NCMM, is that this type of structural biology is very important for drug development. Some improvements are perhaps needed in terms of resolution, but this is definitely one translatable use.
Another exciting area, which I worked on during my PhD, are cellulases and how we can improve them. One of the things we could do is to generate cheaper liquid fuels that are also carbon neutral. One idea is the development of second-generation liquid fuels, and to make ethanol from cellulose. Cellulose is very hard to degrade, because the enzymes that break it down are not very efficient. We could therefore use cryo-EM to better understand the structure of these enzymes, and then see how we might be able to alter them and make them more efficient.
Medical research aside, there are also lots of potential environmental applications that we can use cryo-EM for.
Hartmut Luecke:
The best way to explain how we can use cryo-EM is that 3-dimensional structures of proteins really help to build our understanding and allow us to see how something is put together. Once we have the 3D structure of an object, we can then try figure out how it works. We can see what’s essential, where the active parts are, and how things are functioning.
As far as translational medicine is concerned, structure-guided drug discovery is a great example of how we could potentially use cryo-EM.
With the example of a protein, cryo-EM can help us to find small (organic) molecules that would might fit into a binding cleft. The location of this binding would typically mean that it is inhibiting the target protein, and so you could effectively shut down the function of a protein by binding something that blocks its active site.
You can also try the opposite, finding a protein activator, which is more complicated. This would be trying to fix a broken protein, for example a mutated protein found in many metastatic cancers, by binding something in a certain part that restores its working shape.
What does the future hold for cryo-EM and for Norway?
Hartmut Luecke:
To me cryo-EM is much like crystallography was in the 1980s. Then it was still quite ad-hoc and disorganised, but today it’s very slick and robust with a lot of automation. I think the same will happen with cryo-EM, particularly as more and more people realise its importance and potential. I’m sure it will also become cheaper and more accessible with time, which will also help drive innovation.
I also hope we will be able to strengthen our Nordic collaborations, particularly within the Nordic EMBL Partnership. If possible, we would of course like to join Denmark and Sweden in the CryoNet network of Nordic countries.
We would of course need to meet the terms of membership, and find a way to contribute appropriate resources, as the other members currently do. It would be fantastic to join this network and to benefit from the collaboration and exchange of expertise that such a network offers.
Eva Cunha:
If Norway were to get a cryo-EM microscope, I think it would help to open up a whole new job market. The growth that we’ve seen in places like Denmark, Sweden, and Germany is really exciting, and I think the same would happen here. We would also be really willing to teach people how to prepare their samples, to run the microscope, and to also properly interpret and use the data generated. There will soon be so many more opportunities for working in cryo-EM and through training people here, we can help to prepare them to take advantage of this.
I guess in summary, cryo-EM, both our field, which is single particle cryoEM, and tomography, which allows you to look at larger in situ complexes, really has massive implications. It would be really exciting, not just for NCMM, but for Norway as a whole, to be able to establish a cryo-EM facility here.
We could use it for better understanding human health, environmental matters, agriculture, fishing…the potential is huge and I think that we would be able to contribute a lot to the field.
Poul Nissen shares an overview of how cryo-EM was established in Aarhus, and of the CryoNet network:
Poul Nissen:
Aarhus has always had an electron microscopy facility at Aarhus, but things really picked up in 2008 in terms of cryo-EM.
It all came together around 2012 and 2013 when we got the Titan Krios microscope – one of the first ones in the world. We were lucky to recruit a researcher, Arne Möller, to
DANDRITE, who really got things going and we are really proud that, as a result, DANDRITE has been very instrumental in pushing the establishment of cryo-EM in Denmark forward.
Denmark now has two facilities; one at Aarhus and one in Copenhagen at the Novo Nordisk Foundation Centre for Protein Research.
Having another facility in Denmark has been really useful for us, as we’re able collaborate a lot and help each other out.
We helped them to get the application for their facility going, and a big grant for their facility actually also includes funding for us in Aarhus on a national basis. We’re now also both members of the CryoNet network.
What is CryoNet, and how does it support research?
CryoNet was established in early 2018 following an initiative from the Novo Nordisk Foundation in Denmark and the Wallenberg Foundation in Sweden. These two organisations quickly realised that cryo-EM and its development in Sweden and Denmark was extremely important, and they had the idea of setting up a collaborative network. To do so, they asked us to help organise a Danish-Swedish network for the new Titan Krios facilities that had been established in Umeå, Stockholm, Copenhagen, and Aarhus.
The network is mostly driven by ourselves in terms of what we need to continue pushing cryo-EM forwards. There is an annual meeting, the first of which took place in October 2018, and we also arrange training courses.
We’re very collaborative and open in terms of sharing expertise, and we act as a first line for students who want to learn more about cryo-EM.
As it’s happened, both MIMS and DANDRITE, have been very involved in building up cryo-EM facilities in Umeå and Aarhus. CryoNet is not orientated towards the Nordic EMBL Partnership as such, but it happens to involve two of our centres. The Wallenberg and Novo Nordisk Foundations are very happy about these links, and see the real collaborative potentials here.
Of course, I also hope that Norway is able to one day join the CryoNet network, once facilities are established there. The network is currently based in Sweden and Denmark, so a new partner would be expected to contribute equally in terms of finance, but also understand how the network is set up. It’s very much focused on ‘open, no-strings attached’ science, but I imagine Norway and NCMM would be a great fit for this.