Emma Doughty discusses the potential, and challenges, of cryo-electron tomography
The development of cryo-electron microscopy (cryo-EM) allowed a leap forward in imaging the structure and function of individual biological molecules, an achievement rewarded with a Nobel prize in 2017. Now cryo-electron tomography (cryo-ET) is emerging as a powerful method for performing structural studies in unperturbed environments, i.e. in situ. This offers the potential to study functional modules and their interactions, the molecular sociology of cells, with near-atomic resolution.
Technical developments in structural biology
X-ray crystallography used to be the pre-eminent method for imaging biomolecular structures. This workhorse of the bioimaging world has led to countless discoveries, and will continue to do so. However, X-ray crystallography has a serious limitation – it can only be used to investigate crystallised proteins. Researchers can spend years trying to coax uncooperative proteins into crystals suitable for analysis, and some of the most biologically important molecules are still resisting their attempts. Furthermore, for more complicated biomolecules it becomes increasingly difficult to make useful crystals.
In the early 1980s, Jacques Dubochet developed a technique to ‘vitrify’ molecules by flash-freezing solutions of proteins using liquid ethane, which prevents water-soluble biomolecules from drying out in a vacuum, allows them to retain their natural shape, and keeps them relatively still during electron microscopy, and cryo-EM was born. Since then, improvements in the sensitivity of electron microscopes, in sample preparation techniques and in image-processing software have caused a ‘resolution revolution’, and a corresponding explosion in the number of researchers using cryo-EM.
What does cryo-ET have to offer?
Cryo-ET goes a step further, offering 3D visualisations of molecular complexes in their native, fully hydrated environment, and is therefore ideally suited to reveal cellular organisation at molecular resolution. It’s like a CT scan for cells and biomolecules, using a series of 2D ‘slices’ through the sample to reconstruct a 3D image. The holy grail of in situ structural biology is to obtain subnanometre or even near atomic resolution maps of entire cellular landscapes. Can this be achieved with cryo-ET?
Wolfgang Baumeister researches cellular machines called macromolecular complexes at the Max Planck institute of Biochemistry in Munich. His team developed cryo-ET in the late 1980s, leading to the first successful mapping of macromolecular complexes in intact cells in 2000. For him, one of the main challenges in the uptake of cryo-ET is the start-up costs. He says, “To start a lab that can do cutting-edge research along the these lines, one has to make minimal investment of some something like €10 million. That’s affordable for larger institutions, but there are many other scientists out there who have interesting and challenging problems who cannot make such an investment, and they need to be given access to it and to the technology.”
The solution to the access problem is to create national cryo-EM/ET facilities. In the UK, the Electron Bio-Imaging Centre (eBIC) was established at Diamond Light Source, the UK’s national synchrotron, with the award of a £15.6 million grant from the Wellcome Trust, the Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC). Diamond and eBIC allow scientists to access state-of-the-art cryo-EM facilities, for both single particle analysis and cryo-tomography. Scientists are using onsite cryo-electron microscopes to study everything from complex virus structures to never-before-seen proteins.
Cracking the HIV code
Peijun Zhang is the director of eBIC, where her research focuses on both cryo-EM technology development and investigation of human pathogens such as HIV-1. According to UNAIDS, there were 36.9 million people living with HIV in 2017, of which 1.8 million were newly infected. There is currently no cure or vaccination for HIV, and 35.4 million have died from AIDS-related illnesses since the start of the epidemic. The key to developing a cure for HIV lies in understanding its structure, and how it infects human cells, a complex code that has proved hard to crack.
In 2013, researchers used cryo-EM to view the structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution, and cryo-ET to recreate a 3D structure of a native HIV-1 core. Their work paves the way for further studies of capsid function and for targeted pharmacological intervention. More recently, cryo-EM has shown the structure of Cyclophilin A in complex with the assembled HIV-1 capsid at 8 Å resolution, offering new insights into how the protein stabilises the HIV-1 capsid and is recruited to facilitate HIV-1 infection. The next steps in this research are combining cryo focused ion beam microscopy (cryo-FIB) and cryoET in situ investigations of virus-infected cells.
Structural biology and drug design
Dave Stuart is Diamond’s director of Life Sciences, and a world leader in Structural Biology. His research has included mapping the Foot and Mouth Virus disease structure, discovering the first structure of an enveloped virus, and advancing our understanding of viral assembly, replication, and infection. Known for pushing technological developments that drive innovative science forward, Stuart was a key player in the case for funding Diamond and eBIC.
The technical developments made at synchrotrons have enabled the process of getting structural information on the therapeutic targets and on the complex of a drug with its target to be speeded up orders of magnitude. He says, “It’s speeded up with automation – now you can respond quickly enough in terms of providing structural information to hook directly into pipelines for developing new chemistries. So I think it’s had a fundamental change in the way people think about drug discovery.
The driver for setting up eBIC was to do the same thing for cryo-EM/ET; to provide a centre with enough critical mass to be efficient, to help to try and develop the methods to try and develop the throughput of electron microscopy.
If you collect X-ray diffraction data at Diamond, the software will automatically take it through an analysis pipeline, often to a result, without any intervention. Stuart says: “It takes a long time to set that up – years of work – but once you achieve it then you can open up methods to a much broader community. And the same is starting to be true for electron microscopy. It’s at an earlier stage, the methods are still changing quite rapidly, but one of the challenges that people in Diamond and elsewhere are trying to address is: can we make pipelines so that people can come with a sample, and very quickly get some sort of feedback on the quality of the data and help with analysis of the data?
“It’s clear that there’s still a lot of potential development in the area of electron microscopy, and for me, I think the really interesting possibility – or one of the really interesting possibilities is – to do a different sort of science. It should be feasible, and there’s a lot of technical issues standing in the way still - to join up structural biology with cell biology, so that you can understand what molecules are doing in the very crowded environment of a living cell.”
