Cryo-EM: A machine to shape the future of molecular medicine

ARC Centre of Excellence for Advanced Molecular Imaging

Life and health ultimately depend on the interactions of large biological molecules, such as proteins and carbohydrates. And those interactions depend on the 3D shapes and structures of the molecules.

Although we can gather detailed information on the physical and chemical structure of some of these molecules using X-ray crystallography and can employ computers to suggest their shapes, the range is limited. Many molecules and most molecular interactions are not susceptible to crystallisation.

The only technology that allows us to image the shapes of proteins directly is the transmission electron microscope (EM), in which a tightly focused beam of electrons is passed through a thin sample mounted on a grid-like microscope stage. The image is constructed from data collected on the angles and numbers of electrons deflected and absorbed. But unfortunately, the techniques used to prepare samples for conventional transmission EM tend to distort the shapes of biological molecules.

One way around this is to prepare and study samples that have been snap frozen in clear or vitreous ice. This not only holds in place the shape of molecules and the complexes they form, but it also does so in their natural cellular environment—water. And that’s exactly what a cryo-EM does.

Cryo-EMs have become central to the study of the molecular interactions that drive the functioning of the body, from the normal workings of the immune and nervous systems to what goes wrong in diseases such as cancer and malaria. They can provide us with direct images of the shape and interactions of molecules, how they come together. And this understanding can be combined with detailed structural information gathered using other technologies to form the basis for modelling the course of diseases and for designing therapeutic drugs.

Today, Monash University launches the new FEI Titan Krios cryo-EM purchased with generous support from the Ramaciotti Foundation. It is Australia’s most powerful biological microscope, and will be housed in the new $20 million Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy which will provide the people, expertise and infrastructure to allow users to make the most of its capabilities.

“This sophisticated technology, and the knowledge of the people who will run it, are critical to the success of the ARC Centre of Excellence for Advanced Molecular Imaging and its aspirations of observing how our immune system operates,” says Prof James Whisstock, director of the Centre of Excellence that will be responsible for managing the Ramaciotti Centre. “And through the Imaging Centre, the opportunity to use the Titan Krios will be made available to all Australia’s structural biologists.”

Already Australian researchers are using or proposing to use the cryo-EM to study many molecular interactions, including:

  • drugs that can prevent the spread of the malaria—Dr Wilson Wong, the Walter and Eliza Hall Institute of Medical Research (WEHI);
  • key molecules that help infective bacteria acquire resistance to front-line drugs—Prof Trevor Lithgow, Microbiology, Monash University;
  • the molecules of the mitochondria, the energy powerhouses in all cells—Prof Michael Ryan, Monash University;
  • insulin and its receptor, the key to diabetes, and possibly significant in Alzheimer’s disease and cancer as well—A/Prof Mike Lawrence, WEHI;
  • perforins, the molecules which form pores in the membranes of infected cells as a precursor their elimination—Prof James Whisstock, Imaging Centre, Monash University;
  • receptors that trigger the T cells of the immune system—Prof James McCluskey, The University of Melbourne and Prof Jamie Rossjohn, Imaging Centre, Monash University;
  • transcription, the first step in the process by which genetic material is transcribed by giant enzymes called polymerases—A/Prof Hans Elmlund and A/Prof Dominika Elmlund, Imaging Centre, Monash University.

Access to the cryo-EM technology is now so highly sought after worldwide, that it has become increasingly difficult for Australian researchers to book time at facilities abroad.

Dr Wilson Wong of WEHI leads research that has shown, for the first time, how the drug emetine prevents the malaria parasite, Plasmodium falciparum, from functioning. Using a cryo-EM in Cambridge UK, he and his colleagues collected images of the drug clogging up the parasite’s molecular protein production machinery. It’s a huge step in fighting a disease that still infects hundreds of millions of people and causes more than 600,000 deaths a year.

The researchers were all set to continue their work developing new, better-targeted anti-malaria drugs, but the cryo-EM they were using is already booked solid for years ahead.

The Monash FEI Titan Krios has been secured just at a time when upgrades of its detectors and cameras have provided a biologically significant boost to the microscope’s resolution, says Dr Georg Ramm who will manage the Ramaciotti Centre. Until recently, after passing through the sample, electrons were detected as light scintillations on a screen. Now they are detected directly, using semiconductors. And faster cameras grab and process the information into digital images that are much sharper. Whereas before cryo-EMs could show the overall shape of large molecules, the new Monash Titan Krios can provide important details of loops and side chains in 3D.

And that’s got people like A/Prof Mike Lawrence from WEHI very excited He has been working for several years with a cryo-EM group in Munich using the old light-based camera technology to image insulin receptors and their interaction with insulin. “We have struggled for three years, and I’m still not satisfied with the images we are generating. The new technology will allow us to get sharper images, by a factor of two or three.”

Lawrence is also pleased that the new cryo-EM has been set into a dedicated research centre of people with the experience, expertise and mathematical knowledge to extract the most information from it. “This technology needs to come with a skill base and access regime which allows users to acquire the best data. A cryo-EM isn’t just a turn-key facility. There’s a steep learning curve in applying it most effectively.”

It will add significantly to an extensive and comprehensive suite of technologies for analysing biomolecular structures in the Clayton area of Melbourne. Monash University already operates an extensive range of platform technologies which includes a large X-ray crystallography facility. The Monash Macromolecular Crystallisation Facility which produces much of the material used for X-ray crystallography, for instance is the largest, fully-automated, crystal production facility in the world. Just over the road from Monash is the Australian Synchrotron—the X-ray beamlines of which are used extensively for crystallography.

Monash has made a point of building up its technology platforms. For instance, it also hosts a comprehensive genomics and sequencing facility, known as Micromon; an antibody technology facility and a proteomics facility for identifying and measuring levels of proteins.

All of these facilities are underpinned by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE), a specialised High Performance Computing (HPC) facility for imaging and visualisation. MASSIVE, which is part of the Monash eResearch Centre, provides the computing hardware, software and expertise to drive the latest biomedical research.

“A number of major cross-institutional initiatives, such as the work of the Imaging Centre, are critically dependent on linking several of these platforms,” says Monash Pro Vice-Chancellor (Research and Research Infrastructure), Prof Ian Smith. “They have now been significantly augmented by the new Ramaciotti Centre with its cryo-EM.”

Melbourne is Australia’s most significant centre of structural biology and medical research, and the establishment of the Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy has been supported by a consortium of institutions including Melbourne, La Trobe and Monash universities, WEHI, the Baker IDI Heart and Diabetes Institute, and the Burnet and St Vincent’s institutes of medical research.

In addition to the Titan Krios, says Dr Ramm, the Ramaciotti Centre will house a lower resolution, cryo transmission EM, which will be used to provide preliminary information and data on samples and to assess the most effective ways of employing the Titan Krios. A third EM—a scanning electron microscope—which can form detailed images of the surfaces of samples by detecting the electrons reflected back by them, is also part of the package. In addition, an existing Hitachi transmission EM will be made available, as well as specialised microtomes for taking thin slivers or sections through cells, and other sample preparation equipment.