Monash biomedical facilities: New microscope adds lustre to the cluster

ARC Centre of Excellence for Advanced Molecular Imaging

The launch of the Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy provides a missing piece in Australia’s most extensive and comprehensive suite of technologies to support biomedical research, all based around the Clayton campus of Monash University.

Among the Monash Technology Research Platforms are many focused on analysing the development, activity and structures of biological molecules. Several of these facilities, as well as the Australian Synchrotron just over the road from Monash, support X-ray crystallography, which is the traditional technique used to calculate the structure of protein molecules.

But while it provides detailed structural information, X-ray crystallography has some drawbacks. Less than half of all protein molecules can be crystallised. In particular, larger, more-mobile structures are more difficult to crystallise. Above a certain size—particularly at the level where molecules interact and assemble inside cells—the structures and shapes often have to be inferred from putting together component pieces.

The new FEI Titan Krios cryo-EM, housed in the Ramaciotti Centre, will be able to fill this gap by providing images of exquisite detail, which show the shapes of these complexes and other large biological molecules, ,including in their natural environment inside cells. The Titan Krios can also provide details of changes in shape, molecular interactions and the formation of molecular complexes. This is important when it comes to understanding of the functioning of molecules—particularly when combined with information on structure and activity able to be obtained from the many other Technology Research Platforms.

“A number of major cross-institutional initiatives, such as the work of the Imaging CoE, are critically dependant 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.”

At Monash, for instance, the director of the ARC Centre of Excellence for Advanced Molecular Imaging, Prof James Whisstock is interested in perforin-like proteins which form pores in cell membranes. He and colleagues in Australia and the UK used X-ray crystallography to determine the structure of the repeating units which combine to form the cellular pore.

The immune system employs these perforin pores as conduits for delivering large toxic molecules and nanoparticles into infected cells. They could equally be used to smuggle compounds into cells for therapeutic purposes. So James and his team now want to make different kinds of these molecules, and study the 3D structure of the pores they form under the cryo-EM.

Working alongside James’ lab, research fellow Dr Michelle Dunstone and her team has been studying another perforin-like protein, pleurotolysin, in oyster mushrooms. She and her UK collaborators have already been using a cryo-EM in Britain to image different stages of pleurotolysin pore formation. But while the cryo-EM is significant to the work, it is only one of a series of the Monash platform technologies Michelle has been using. Being able to combine several such technologies is critical to such work.

Michelle has drawn on the Micromon facility for DNA sequencing, the Monash Protein Production Unit to help make the protein, and the Monash Biomedical Proteomics Facility to assess the quality of the protein. The automated technology of the Monash Macromolecular Crystallisation Facility helped her prepare her protein for X-ray crystallography studies, some of which were undertaken using that facility’s in-house X-ray sources. And some early imaging was done with the assistance of Monash Micro Imaging, before sending samples to Britain for the high-end cryo-EM work. Finally, interpretation of the data would have been impossible without the computer resources of Monash University’s visualisation environment, known as MASSIVE, and the super-computers of the Victorian Life Sciences Computation Initiative (VLSCI).

“The FEI Titan Krios at Monash is the start of a new era for structural biology in Australia,” Michelle says. “Using it, we will be able to see the shapes of proteins clearly, and how those shape as they change as they function. The new Centre will provide a central hub into which information from other research technologies can feed.”

In addition to the cryo-EM, Monash facilities relevant to molecular biology include:

  • Micromon—which provides DNA sequencing by means of two different technologies;
  • Monash Protein Production Unit—which specialises in the production and purification of specific proteins for research;
  • Monash Antibody Technologies Facility—one of only two facilities in the world that produces custom made, high affinity rodent antibodies against proteins and peptides;
  • Monash Biomedical Proteomics Facility—which is able to identify and measure the concentrations of even rare proteins by combining data from four mass spectrometry systems;
  • Monash Macromolecular Crystallisation Facility—which houses one of the world’s largest fully automated systems to determine how to crystallise biological molecules, particularly proteins. It also houses X-ray diffraction equipment;
  • Monash Animal Research Platform—which breeds, supplies and cares for specialised laboratory animals, particularly rodents;
  • FishCore—which is the largest zebra fish facility in the southern hemisphere. Used for studies to do with animal embryology and development;
  • FlowCore—where high speed, laser-based technology is employed to identify, count, sort and measure cells;
  • Monash Histology Platform—where biological samples are prepared and stained for microscopes—in paraffin or resin, at room temperature or frozen; and
  • Monash Micro Imaging—a cluster of facilities which provide access to many different forms of microscopy, both light, including super resolution, and scanning and transmission electron microscopy

All the above facilities area underpinned and supported by:

  • Monash eResearch Centre—which specialises in the collection, storage, processing and management of research data. Its instrumentation includes the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) which is particularly useful for imaging, modelling and simulation.

What’s more, just over the road from Monash Clayton is the Australian Synchrotron. Several of its beamlines are of particular interest for biomedical research. Combining crystallography with small angle X-ray scattering (SAXS), for instance, provides valuable insights into the behaviour and tertiary structure of large biological molecules in solution.