Zooming in on high-quality crystals from FlowCore
We’ve come a long way since Rosalind Franklin used X-ray diffraction to study crystals of possibly the world’s best-known biological molecule: DNA. These days, microfocus beamlines at synchrotrons can focus in on the ultra-fine details of molecules such as micrometre-sized proteins – that’s one-thousandth of a millimetre across.
Traditionally, such molecules have been crystallised before study using in vitro techniques (i.e. in a glass flask). Until now, the cells had to be broken open and the crystals purified separately – a complicated, inefficient and time-intensive process that often gives poor-quality results. Researchers can also now study in vivo protein crystals, which form naturally inside a cell.
Using the laser-based technique of flow cytometry, FlowCore – one of the Monash Research Technology Platforms – has solved this bottleneck in protein structural analysis.
Instead of breaking cells open to purify the protein crystals, the living cells can be sorted into two groups: cells that contain crystals and those that don’t. The technique also gets rid of any broken cells and debris that might clutter up the sample.
The result? A high-quality, clean, concentrated sample of cells full of the protein of interest. Once purified to this level, the cells are stained and spread onto a gridded mesh support. The support can then be taken to the X-ray beam, where the cells – being much larger than a purified crystal – can be easily aligned with the beam and focussed upon.
Another advantage to using whole cells is that the protein crystals are maintained in the ‘cellular context’. In other words, the technique doesn’t disrupt any interactions the protein of interest might have with other proteins or different molecules, giving a more biologically relevant result.
Researchers from Monash’s Structural Virology Laboratory recently used FlowCore’s lab to purify crystals of a silkworm viral protein grown inside insect cells. They found that this streamlined method produced much higher-quality samples, which they analysed at the Australian Synchrotron. The data they obtained were of much better quality than would have been possible using conventional methods.
With four high-speed sorters at the moment, but the capacity to expand to six, FlowCore is one of Australia’s top flow cytometry facilities. Manager Andy Fryga is proud of the centre’s ability to help researchers from a wide range of fields: whether from commercial, academic or medical backgrounds.
“We’re challenged by lots of different researchers – from microbiology to ecology and environmental biology,” he says. “Our goal is to say ‘yes’ more times than we say ‘no’! We can do this because we have very high-end equipment, and the best possible staffing structure.”
But for Fryga, it’s not just about the best and latest equipment. He’s just as keen on helping people get the most out of FlowCore’s services.
“The technology is actually quite daunting; we try to break it down and make it more accessible,” he explains. “We want researchers to not worry so much upfront about the technology. We’re here to take them on a journey: and hopefully, at the end, they’ll be better researchers.”