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LMB unveils vision for next generation of Nobel Prize-winning cryo-EM technology




It has revolutionised how biological molecules are imaged, enabling scientists to resolve structures that were previously beyond reach.

The impact of cryo-electron microscopy, or cryo-EM, was underscored by the award of the 2017 Nobel Prize in Chemistry to three scientists who played crucial roles in its development – including Richard Henderson, of the MRC Laboratory of Molecular Biology (LMB) in Cambridge.

Chris Russo at the MRC LMB with the detector. Picture: Keith Heppell
Chris Russo at the MRC LMB with the detector. Picture: Keith Heppell

Cryo-EM, however, remains beyond the reach of many laboratories.

For a start, the machines are prohibitively expensive – one can set you back around £5million and annual running costs are in the region of £250,000.

And they are huge: weighing in at about a tonne and several metres tall, these are no ordinary microscopes.

But now the LMB – which has three such Titan Krios microscopes among its seven cryo-EM machines – has unveiled a vision for the future of the technology, making it more accessible, 10 times more affordable and requiring a fraction of the space.

Chris Russo’s group at the LMB used currently available technology to assemble a microscope that demonstrated the potential.

A 3D reconstruction of DPS protein at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology
A 3D reconstruction of DPS protein at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology

The key, they found, is in reducing the electron energy in the cryo-EM from the current standard of 300 or 200 kiloelectron volts (keV) to 100keV.

Based on previous work by Chris, Mathew Peet and Richard Henderson, they believe 100keV not only simplifies the instrument but is the optimum voltage for maximising the amount of data collected, while taking into account the radiation damage caused to the biological samples by the electron beam.

Chris told the Cambridge Independent: “The real surprise here is that lowering the energy of the electron beam in the microscope not only makes the instrument cheaper, but will likely make it better as well. It’s a rare win-win that the physics dictates a way to make things cheaper and better at the same time.”

Working out the structure of biological molecules is crucial for understanding their function and interactions, and is key to developing new drugs.

From left, Katerina Naydenova, Greg McMullan and Chris Russo at the MRC LMB. Picture: Keith Heppell
From left, Katerina Naydenova, Greg McMullan and Chris Russo at the MRC LMB. Picture: Keith Heppell

For a century, X-ray crystallography was the technology used for this purpose, but it is limited by the need to turn samples into crystals before they can be studied.

Some large molecules and complexes of molecules will not form crystals, and many proteins important in disease are particularly difficult to crystallise.

With cryo-EM, there is no need to form crystals. Instead, samples in an aqueous solution are applied to a grid mesh and plunged into a bath of liquid ethane.

This quickly vitrifies the water around the sample, turning it into a disordered glass, rather than crystalline ice, which would absorb the electron beam and obscure the sample.

Accelerated electrons are then fired through this very thin sample. These tiny charged particles have such a minute wavelength that they can interact with nanoscale components, unlike the photons in light used in standard microscopes.

Two-dimensional images are collected, from which a 3D shape can be built up. Near-atomic resolution can be achieved and imaging at cryogenic temperatures reduces the radiation damage to the biological samples.

A 3D reconstruction of E. coli 70S ribosome at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology
A 3D reconstruction of E. coli 70S ribosome at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology

One drawback of the technique is that it has a low signal-to-noise ratio, although averaging multiple images can help to overcome the problem.

Chris’s group experimented with reducing the voltage of a standard, commercial 200kV field emission gun (FEG) microscope to 100kV, then attached a commercial hybrid-pixel camera designed for X-ray detection to the camera chamber for low-dose data collection.

In one week, they imaged five single particle specimens – hepatitis B capsid, bacterial 70S ribosome, catalase, DNA protection during starvation (DPS) protein and haemoglobin – which range in size.

A 3D reconstruction of hepatitis B virus capsid at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology
A 3D reconstruction of hepatitis B virus capsid at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology

The data was used to construct 3D structures with resolutions between 8.4 and 3.4 ångstroms (Å). One ångstrom is equivalent to one 10 millionth of a millimetre or 0.1 nanometers.

“More than 90 per cent of the structures solved on all the high end cryo-EM microscopes worldwide in the last year were in the 9-3Å range, which is exactly what we demonstrated with this prototype,” said Chris. “So this new microscope would be appropriate for more than 90 per cent of the work currently under way and potentially be 10 times less costly.”

Current cryo-EM machines are much more expensive because of the need for X-ray shielding and high voltage power supplies.

The technology being developed by Chris’s group could be housed in a room about one-sixth the size.

A comparison of the room requirements for cryo-EM machines with different voltages Image: MRC LMB
A comparison of the room requirements for cryo-EM machines with different voltages Image: MRC LMB

In addition to slashing the price of the microscope, set-up costs would also be dramatically reduced from around £500,000 to £50,000. Running costs would be greatly cut back to around five per cent of the current £250,000 a year.

“Still, more work needs to be done to turn this lab prototype into a commercial microscope that any biology lab can buy,” said Chris.

“The main limitation now is the detector – the prototype used a small camera that is similar in number of pixels to an early mobile phone camera.

“We have demonstrated the potential but now we need bigger, faster and more powerful electron cameras for this to really take off.”

A 3D reconstruction of haemoglobin structure at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology
A 3D reconstruction of haemoglobin structure at 100 keV. Image: Chris Russo's group at the MRC Laboratory of Molecular Biology

Greg McMullan led the work to demonstrate the use of a hybrid-pixel detector to image electrons. The detector used was 32 times smaller than that used in a commercial Titan Krios cryo-EM machine, as the lower voltage leads to much greater electron scatter.

A solution would be to develop a design using a larger pixel, which matches the range of electron scattering in the detector.

“Based on this work, several companies and research groups are now working towards this goal and hopefully in a few years this technology will be widely available, as part of a simple cheap microscope that any biology lab will be able to afford,” said Chris.

The LMB aims to help construct a full prototype microscope based on the principles and specifications set out in the paper.

The work – funded by the MRC, the Cambridge Commonwealth, European and International Trust and a Bradfield scholarship – forms part of the LMB’s drive to improve the accessibility of cryo-EM for scientists around the world, accelerating its impact in biology and helping to improve human health.

Electron cryotomography, a related technique for imaging thick specimen and cells, would still require higher energy instruments.

Cryo-EM has already transformed the imaging of biological samples. In its early days, it took about a year for the LMB’s Tony Crowther and others to resolve the hepatitis B virus capsid structure to 7.4Å resolution in 1997.

A cryo-electron microscope
A cryo-electron microscope

Chris’s group resolved the same structure to 8.2 Å based on a few micrographs in less than an hour.

With this latest proof-of-principle study, the next generation of cryo-EM has taken a big step nearer.


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