The Centre has collaborated with a global business to add value, accelerate innovation and provide crucial validation of prototype techniques.
For two weeks, Imaging CoE postdoc Chris Lupton and Dr Hervé Remigy, applications engineer of the Life Sciences Business Unit of FEI Company, combined their expertise to test a new approach to optimise samples prior to preparing them for vitrification and subsequent cryo-transmission electron microscopy (cryo-EM).
FEI Company has been developing an automated ‘walk away’ system, ProteoPlexTM, to optimise the buffer conditions that are critically important in obtaining a homogenous and well-dispersed isolated protein suspension. After all, an optimal specimen determines the quality of the final 3D image reconstruction by applying single particle analysis – the main application within cryo-EM.
The overall goal of sample preparation is to feed the analytical technique of choice with protein subsamples that maximise the chance of successful analysis. Currently, choosing an appropriate buffering mixture is a game of estimation, approximation and chance.
“ProteoPlexTM can automatically screen a wide range of buffering conditions and determine the optimal pH and buffer to stabilise any multiple-protein complex,” explains Dr Remigy. “And once the optimal buffer has been found we can then use ProteoPlexTM to automatically screen various ligands to find other molecules that may further stabilise the complex.”
Building on recent advances in differential scanning fluorimetry, FEI’s algorithm is so sensitive it will reliably rank different conditions of a certain well, impossible to do using traditional methods of analysis. This helps to mitigate imaging protein complexes that have disassociated or aggregated.
“Imaging multi-protein complexes is a difficult challenge. Being able to do this with less, or no, aggregation of the complexes will really assist in structure determination experiments. If each protein in the sample is distinct, it makes image analysis easier and more efficient; it also means that we only have to consider differences in orientation, rather than variability brought about by poor sample quality,” says Lupton.
Dr Marc Storms, Product Marketing Manager of the FEI Business Unit Life Sciences, says many strategic benefits and scientific insights have been gained and learnt through the collaboration.
“The Centre of Excellence in Advanced Molecular Imaging at Monash University is a strategically important site for FEI,” he says. “As one of the leading centres for a wide variation of imaging technologies, the CoE is also spearheading in the adoption and developments of cryo-TEM. As FEI is further intensifying its workflow philosophy (both up and downstream), we would like to adopt as much hands-on knowledge in the areas of both sample preparation and image handling and processing. Monash is the perfect incubator for this,” concludes Dr Storms.
Dr Remigy tells us that the environment provided by the CoE triggered the decision to start this collaboration at the Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy. “FEI’s aim to provide researchers with a new technique and tool to inform decisions has been aided significantly by working with the researchers here in the Imaging CoE. We have also gained unique insight into how our tools are used and just how useful this new one will be,” he says.
Through this partnership, Imaging CoE investigators are being trained in state-of-the-art techniques while validating and improving them. Projects like this do not come up very often, and being engaged by FEI is testament to the Centre’s outstanding research track record.
“Now a comparison between cryo-EM images of ProteoPlexTM optimised and non-optimised samples is required to assess the level of improvement provided by this new method,” concludes Dr Remigy.
Over the two weeks, Dr Remigy and Lupton painstakingly pipetted 11 protein complexes and the algorithm automatically determined optimal buffer conditions for each thermal titration assay – three or four look really promising.