What exactly is cryo-EM, and how does it work?

As Poul Nissen explains, X-ray crystallography has been used for decades to obtain information about protein structures. While this is still an important technique, the main difference when it comes to single particle cryo-EM is that, rather than X-ray photons, cryo-EM uses electrons, which interact much more strongly with matter. Advances in technology mean that researchers are now able to expand upon the level of detail that they can extract – it’s now possible to see the structures of individual molecules or macromolecules.

As Poul Nissen explains: “We can take a sample as it is without having to generate crystals, as is required in X-ray crystallography, and make very thin deposits on a grid. This is then quickly frozen in liquid ethane. The freezing creates a very thin layer of vitreous ice which we can look through using a cryo-electron microscope, and then see the individual proteins, or macromolecules, in the sample in their flash cooled state. 

"Thanks to the level of detail we can extract, we’re now able to get a direct insight into the atomic structures that we’re examining,”

What are the main advantages of using cryo-EM?

As Hartmut Luecke, Acting Director of NCMM, describes, one of the main benefits of using cryo-EM is that sample preparation is a lot more straightforward, 

“You can get away with using a much smaller amount of sample and most importantly, you don’t need to crystallize it." 

"The vitrification process is also another advantage of cryo-EM – your sample is your target protein in solution frozen in time, so the contents are as close in terms of appearance to their pre-frozen state as possible.”

What is the current cryo-EM set-up within the Nordic EMBL Partnership centres?

Aarhus has had electron microscopy facilities for decades, but things picked up for cryo-EM in 2008. 

Poul Nissen: “We recruited the first cryo-EM researchers in 2008, and around 2011 we got the Titan Krios microscope – one of the first ones in the world. Later, we were fortunate also to recruit a cryo-EM team leader at DANDRITE, Arne Möller, who helped to drive this together with Thomas Boesen, a DANDRITE affiliated researcher and now cryo-EM facility manager. 

"We are proud that, as a result, DANDRITE has been very instrumental in pushing the establishment of cryo-EM in Denmark forward.”

Denmark currently has two cryo-EM facilities; one in Aarhus and one in Copenhagen at the Novo Nordisk Foundation Centre for Protein Research. Poul Nissen continues, “Having another facility in Denmark has been useful for us, as we’re able to collaborate a lot and help each other out. For example, we helped them to get the application for their facility going, and a big grant for their facility also includes funding for us in Aarhus on a national basis. Together we have raised national funding to strengthen facilities for a general user community in Denmark. Both sites are also members of the CryoNet network.”

Umeå University has also had a cryo-EM facility for some years. The work to develop a facility was initiated in 2014 after the essential funding was awarded from The Knut and Alice Wallenberg, and the Kempe Foundations. These two foundations provided support based on a proposal from Umeå Centre for Microbial Research (UCMR), drafted together with other colleagues at several Swedish universities, for a Cryo-EM platform as a national resource in Umeå. It is now part of The Cryo-EM Swedish National Facility offering access to state-of-the-art equipment and expertise in single particle cryo-EM and cryo-tomography (cryo-ET) at the two nodes; at SciLifeLab in Stockholm and at Umeå University.

Bernt Eric Uhlin, also Founding Director of UCMR, explains how the facility has evolved: 

“In 2009 some scientists affiliated with MIMS and UCMR suggested that the new developments in cryo-EM technology would be beneficial for our infection biology research. The 2011/2012 arrival of Dr. Linda Sandblad, as Assistant Professor funded by UCMR, was a key recruitment for such efforts. 

"Linda, who did her PhD work at EMBL, came with expertise in cryo-EM-tomography and soon took responsibility for the EM platform development in Umeå. She is now our SciLifeLab Head of Facility for Cryo-EM and the Director of Umeå Core Facility for Electron Microscopy (UCEM)."

"Whereas I am acting as Facility Director jointly with Gunnar von Heijne at SciLifeLab, of the National Facility, my personal experiences of the technique are still relatively limited although my lab currently is pursuing such studies and has published cryo-EM data on bacterial structures in a collaborative study.”

What is the CryoNet network?

CryoNet was established in early 2018 following an initiative from the Novo Nordisk Foundation in Denmark and the Knut and Alice Wallenberg Foundation in Sweden. 

Poul Nissen: “These two private foundations 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 regarding 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 facility staff meetings jointly and training courses. 

"We’re very collaborative and open regarding sharing expertise, and we act as the first line for students who want to learn more about cryo-EM.”

MIMS and DANDRITE have both been very involved in building up cryo-EM facilities in Umeå and Aarhus, although CryoNet is not explicitly oriented towards the Nordic EMBL Partnership per se. 

Poul Nissen continues: “I hope that Norway is also able to 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 regarding finance, but also understand how the network is set up. It’s focused on ‘open, no-strings-attached’ science, but I imagine Norway and NCMM, and in fact also FIMM and Finland, which has already long had a very strong cryo-EM community, would be an excellent fit for this.” 

What are the plans to introduce cryo-EM to Norway?

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. This means that researchers like Hartmut Luecke and his group have to travel to use existing facilities in Umeå and Frankfurt, which can be challenging given the logistics involved with transporting samples. 

However, Professor Luecke, is hoping that, pending a successful joint funding application with partners at the University of Oslo and other Norwegian universities and hospitals, Norway will be able to establish its own cryo-EM facility:

“The main challenge, as with setting up any 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 Radiumhospital, UiO; NCMM and the universities of Bergen, Tromsø, and Trondheim have recently applied for an infrastructure grant from the Research Council of Norway as part of a wider national consortium. 

“If successful, we will install an Arctica cryo-electron microscope here in Oslo in late 2019. The dream would, of course, be to also have a Titan Krios microscope in the future, as researchers have at DANDRITE and MIMS. The resolution that these produce is better, but they require a much larger budget.” 

As Professor Luecke explains, having such a facility in Norway would also benefit both his and Norway’s research environment: 

“We have been approached by several people who would like to collaborate with our group, and I think having the proper cryo-EM instrumentation would help drive this forward. It would be great to open up the facility for use by other researchers, and train them in cryo-EM – much like a standard core facility."

“I also think that bringing cryo-EM to Norway would help to drive more successful ERC grant applications, and help with securing more high-profile publications. To have a thriving cryo-EM community here would also mean we could take the lead on helping to train some of the future stars of structural biology, and help to further innovation and collaboration in the Nordics.” 

Why is cryo-EM so important for basic molecular research?

It would appear that cryo-EM, and all of the work around it, is helping to advance basic research. It also appeals to a much broader scientific community. “Another thing that we can now do is examine subcellular features and structures via so-called cryo-electron tomography (cryo-ET). 

"Rather than having a suspension of purified molecules, you can look at a thin slice of cell or tissue, or a reconstituted system, and then observed them in the electron microscope through a tilt series so that the sample is examined from different angles and can be reconstructed in 3D."

"This can then, albeit at a lower resolution, provide cellular landscapes of molecular networks captured in their living state,” says Poul Nissen. 

How can cryo-EM be used in translational medicine?

As Hartmut Luecke describes, the best way to explain how we can use cryo-EM is that by seeing the structure of a protein we can understand more about how it works, which is vital for drug discovery.

“In these 3D structures, 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."

"cryo-EM can help us to find small (organic) molecules that would 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.”

There are also applications for connecting cell biology and genetics, according to Poul Nissen: “In genetics, we get all these markers, such as which genes, and which proteins and regulatory mechanisms are essential. With imaging and cell biology we get to see where things are relative to each other. Using cryo-EM, we can put these all together and understand more fully how the molecules interact and lead to the genetic coupling that we observe. 

“Right now, we’re in a stage where we need to build the capacity and expertise of the next generation of scientists – this is vital if we are to progress. In a few years, I’m sure we will have many excellent group leader candidates, also from our own backyard, who will want to come back and set up their own dedicated cryo-EM groups in the Nordics.” 

What does the future look like for cryo-EM in the Nordics?

Poul Nissen: “I think we can be real frontrunners in applying and integrating cryo-EM strongly into molecular medicine. 

"I think we can also really open the eyes of the rest of Scandinavia and show them that this is the way to go and that we already have excellent facilities and expertise here."

He continues: “The cryo-EM facility at MIMS is a great example of a very well set up facility. Their research invites both approaches that I mentioned before; the single particle approach and the tomography approach. Bacterial cells are a great fit for cryo-EM, due to their size, so it works well for MIMS’ focus on molecular infection research.”

The same can also be said for the facility in Aarhus. Researchers at DANDRITE are very much interested in cell-cell interaction networks in the brain, and how membrane proteins interact and allow neurons to communicate; also an excellent fit for cryo-EM.

Bernt Eric Uhlin adds: “With the current developments and establishment of new cryo-EM facilities in our research environments, I think there is good potential for growth in the use of cryo-EM approaches locally and in collaborations throughout the Nordic EMBL Partnership. It would be in line with the aims of the agreement between The Partnership Institutes and EMBL, for example, that we will facilitate each other’s access to their facilities, instrumentation, and databases.

“I think we already have seen a quite rapid movement during the recent few years that most certainly will continue, as the technology will be increasingly accessible as an important approach to more scientists.”

Cryo-EM could also be a way to more closely connect researchers across the Nordic countries.

Hartmut Luecke: “I hope we would be able to strengthen our Nordic collaborations, particularly within the Nordic EMBL Partnership. If possible, we would, of course, hope to join Denmark and Sweden in the CryoNet network of Nordic countries. We would 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.”

Poul Nissen concludes, “The Nordics are an exciting case in this area of research. We are all very closely connected, and also have a strong tradition for good molecular medicine research and registries. These registries and databanks will become very important, particularly when we want to take cryo-EM to the next level – for example when we start analysing patient materials and examining protein structures from actual patients. This is beginning to happen now in patients; such as in neurodegenerative disorders. We’re able to look at the misfolded proteins in tissue samples, and see how this misfolding happened. I think this is quite amazing! 

"I think the set-up we currently have, which will hopefully only grow, means that the future of cryo-EM in the Nordics is undoubtedly very bright. We have the potential to become real leaders in the field."

Further information:

Read a more in-depth interview with Hartmut Luecke, and researcher Eva Cunha, about the case for cryo-EM in Norway on the NCMM website.

Learn more about the cryo-EM facility at Aarhus University.

Learn more about the cryo-EM facility at Umeå University.

Learn more about the CryoNet Network.