Scotland shakes up cryo-electron microscopy


Rebecca Pool

Monday, February 25, 2019 - 12:30
First experimental data from the JEOL 300 ARM at SCMI, following automated collection of a small dataset on this capsid produced by a mutant Herpes Simplex Virus (7 angstrom resolution).
A collaboration between Scotland-based universities is set to make waves in the world of cryo-electron microscopy.
Launched in September this year, the Scottish Centre for Macromolecular Imaging (SCMI) comprises the universities of Glasgow, Edinburgh, Dundee and St Andrews, and will soon be home to myriad researchers intent on understanding life at an atomic level.
But while like-minded centres around the world - including the Francis Crick Institute, Korea Basic Science Institute and New York Structural Biology Center - rely on the Titan Krios cryo-EM to unravel biostructures to ever-greater resolutions, SCMI is different. Its director, Dr David Bhella, and SCMI colleagues, opted for a JEOL 300 ARM.
"With this decision, we are taking Scotland in a completely different direction from the rest of the world of structural biology," he tells Microscopy and Analysis. "My first reaction was to buy the Krios as we know it delivers good quality data, but we looked at this long and hard, visited JEOL's prototype in Japan, watched a flawless demonstration and were so impressed."
Based at the University of Glasgow, SCMI also houses a second JEOL instrument, a 200kV 'F2' Cryo-TEM with a direct electron detector. According to Bhella, this instrument, and two additional 200 kV instruments - a Tecnai F20 at Edinburgh and JEOL 2200 at Dundee - will support the top-end JEOL 300 ARM.
"We wanted to avoid putting a lot of material through the more expensive ARM 300 that would not yield publication quality data," he says. "So, we have these three 'feeder' sites with 200 kV instruments, that will provide technical support as well as equipment to prepare cryo-TEM grids ready for the ARM 300."
Right now, the electron microscopes at the Universities of Dundee and Edinburgh use CCD cameras, but Bhella is hopeful that these instruments will soon be fitted with direct detectors, just like the Glasgow University cryo-EMs.
And researchers will be allocated time on the JEOL 300 ARM according to a peer review system, as takes place with the Titan Krios at the recently opened Electron Bioimaging Centre, eBIC, at UK synchrotron, Diamond Light Source.
At the time of writing, SCMI's leading cryo-EM was in advanced stages of commissioning. The software set-up had been demonstrated, automated single particle and automated tomography had been implemented into the single particle workflow and the centre was very close to introducing its first external projects.
"A penalty of being an early adopter is you have to go through the process of learning to install the microscope at a customer site with the manufacturer," points out Bhella. "But we have already got our first structures from the microscope and are happy that it's performing, so will now ramp up slowly."
"I hope that by Summer [2019] we'll be offering access to anyone with a competent request," he adds.
Bhella's field of interest is virus structures as well as virus structure host interactions, so he expects, alongside colleagues, to be looking at how viral capsid proteins interact with host receptors and neutralising antibodies for vaccine design. "We'll also be solving a number of Zika virus structures with antibodies," he says.
Bhella and colleagues recently imaged Macrobrachium rosenbergii nodavirus - a pathogen of freshwater prawns - using a Thermo-Fisher Titan Krios at the UK Electron Bio-Imaging Centre (eBIC). A total of 40,883 particle images were used to calculate a reconstruction with an overall resolution of 3.3 Å (a to c). Fine structural details of the map were also revealed (d). [Ho KL, Gabrielsen M, Beh PL, Kueh CL, Thong QX, et al. (2018), PLOS Biology 16(10): e3000038.] 
Researchers have access to FIB SEM with a cold stage, so in-situ analyses will ensue. "We're keen to push sample preparation as well as imaging technologies so will study viruses within cells, in-situ," adds Bhella.
Researchers will also be investigating RNA-dependent RNA polymerases as well as DNA-binding proteins and nucleosome structures.
"I'm sure we'll also be seeing lots of ribosome structures," laughs Bhella. "But basically, we'll be looking at anything that's life; infectious disease, cancer biology and fundamental cell biology - we'll be addressing all of this in abundance."
So as SCMI edges close to getting its somewhat controversial choice of cryo-EM fully up and running, how has the rest of the community responded to Bhella's decision? In short, very well.
As Bhella points out, his choice of instrument can only stimulate competition between the key cryo-EM manufacturers - Thermo Fisher Scientific and JEOL - and help to ensure good customer service levels while encouraging innovation.
"The response from the cryo-EM community has been overwhelmingly positive and now I see a kind of impatience from our colleagues to see what we are going to be doing at SCMI," he adds.
And given the swelling number of centres housing cryo-EMs in the UK, and worldwide, the future looks bright for structural and cell biologists. Bhella expects rising automation will see cryo-EM being increasingly used in structural biologists' workflows while FIB SEM and other cryo-sectioning methods will bring more cell biologists on board.
"I think in the next ten years when we've all gotten over the excitement of sub-two angstrom biology, we're going to be very excited about structural, in-situ biology," he says. "It's going to have a huge impact in the life sciences and alongside fluorescence, super-resolution and X-ray microscopy will provide a lot of structural information about the cell."
Why choose the Cryo ARM 300 instead of a Titan Krios?
SCMI Director, David Bhella, cites several reasons for his choice of cryo-electron microscope. 
First, he was 'particularly impressed' with the Cryo ARM 300's automated specimen exchange system.
The system comprises a specimen stage to keep samples at liquid nitrogen temperature and a cryo-transfer system to automatically transfer and store these cooled samples in the autoloader storage area. The system can store up to 12 samples, and importantly, arbitrary samples can be exchanged while remaining samples remain cooled.
SCMI Director, David Bhella.
"A huge part of the functionality of these high-end microscopes is the auto-loader... and the design of this in the ARM is really nice," highlights Bhella. "In the Krios you load twelve grids and after your session bring them all back out again... so the ARM is more flexible here."
Bhella was also drawn to the instrument's cold field emission gun. Cold FEGs are known for producing a high-brightness electron beam with a small energy spread, offering high coherency, and providing high resolution and high contrast imaging.
"The Schottky gun in the Krios is proven to deliver structures to 1.5 angstrom," points out Bhella. "But this should be attainable on the cold FEG and we'll hopefully see other benefits in terms of contrast."
The Cryo ARM 300 is also equipped with an in-column Omega energy filter, which Bhella believes is more convenient to use than the post-column design energy filters. And the SCMI director also selected a DE 64 for the instrument rather than a Gatan K3, which is commonly used with the Titan Krios.
"I love the ThermoFisher Falcon 3, but that is not an option for a JEOL microscope. We have been working with Direct Electron previously and I felt the DE 64 was more attractive for tomography as well as collecting single particle datasets across large viruses," he says.
"Overall, we now have such a well-engineered microscope here," he adds. "I can't say right now if we have made the right decision but this is one of the first four instruments worldwide and JEOL is really invested in helping our centre to work." 
Meet the funders
SCMI secured more than £5 million of funding to create its innovative structural biology centre in Scotland.
This investment is part of £11.3m in government funding, awarded through the Medical Research Council (MRC) to boost structural and cell biology research.
Additional support has come from the SCMI consortium partners (Universities of Glasgow, Edinburgh, Dundee and St. Andrews), Scottish Funding Council, Scottish Universities Life Sciences Alliance (SULSA) and the Beatson Institute for Cancer Research.
A further charitable donation was made by the M J M Smith Trust for the supply of essential computer equipment.
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