New cryo-EM frontier opens at -260C thanks to research at MRC Laboratory of Molecular Biology in Cambridge
Another important advance in the development of electron cryo-microscopy has been achieved at the MRC Laboratory of Molecular Biology in Cambridge – and it could enable the structures of important small membrane proteins to be discovered for the first time, writes editor Paul Brackley.
Chris Russo’s group, in the LMB’s Structural Studies Division, has solved a challenge that has baffled scientists for four decades – following experiments conducted at -260˚C.
Electron cryo-microscopy, or cryo-EM, has become an increasingly valuable tool for mapping the structures of biological molecules.
Samples are rapidly frozen in a thin layer of ice using liquid ethane, loading them onto a mesh-like grid to provide a stable support surface and scanning them with an electron beam.
But the determination of structures at atomic levels has always been limited by radiation damage. Over 40 years, numerous attempts have been made by scientists to reduce the effects of radiation damage by cooling specimens with liquid helium, as this is much colder than liquid nitrogen.
The lower temperature restricts the diffusion of damaging free radicals, reducing radiation damage, but all attempts using liquid helium have so far failed to produce any improvement in single-particle structure determination.
Now, the LMB team has identified the physical causes behind the loss of information in these previous attempts to use liquid helium and they have designed new equipment to overcome the problem.
The result is that they have been able to determine structures using liquid helium cooling where every frame contains more information than that available using liquid nitrogen.
“There are numerous small membrane proteins whose structure cannot be determined by any method – – they are too small for current cryo-EM and they cannot be crystallised,” Chris told the Cambridge Independent, referring to the two methods used to determine biological structures.
“The lack of experimental structures also means that they cannot be predicted accurately. These structures are of great interest in biology and medicine since they are often the gatekeepers and regulators of cell function in health and disease.
“Improving the signal in cryo-EM images by cooling the microscope to even lower temperatures may be the key to unlocking all these structures.”
When samples are cooled using liquid nitrogen, they are taken to a temperature of about 80 Kelvins (K), which is the equivalent of roughly -193˚C.
But liquid helium is substantially colder – it cools specimens to a temperature of 13K, or approximately -260˚C, inside the microscope.
Cooling crystals from target molecules at liquid helium temperatures is known to halve the effects of radiation damage in comparison to liquid nitrogen.
So if current cryo-EM methods could be adapted to use liquid helium, this would increase the information captured – known as the signal – and reduce unwanted variations or disturbances, known as noise.
Joshua Dickerson, a PhD student and then a postdoc in Chris’s group who is now a postdoc at the University of California in Berkeley, wanted to understand the physics behind the loss of data quality seen in liquid helium cooled cryo-EM.
He found the principal cause was that the specimen was moving. Blurriness and loss of spatial detail occurred even if the specimen moves just a few Ångstroms. One Ångstrom is a hundred-millionth of a centimetre.
At liquid nitrogen temperatures, using grids with small holes of 200 to 300 nanometres in diameter (one nanometre being one billionth of a metre) was sufficient to eliminate any movement caused by the electron beam. But at liquid helium temperatures, image quality was compromised. The group sought to understand why by examining the movement of gold nanoparticles in 300nm diameter-holed grids using identical imaging conditions at both 81K and 13K.
They found that at 81K there was no significant particle movement and the grid and the layer of frozen water covering it remained flat throughout.
But at 13K the particles travelled away from the centre of the grid holes – and the rate of displacement increased as hole diameter increased.
At a temperature of 13K, stress accumulated in the grid as the layer of frozen water contracted at differing rates, which caused the grid to crinkle. When the specimen was exposed to the electron beam, the frozen water became an ultra-viscous fluid, which allowed the grid to relax and resume its flattened shape.
But the group was surprised to find that when the 13K specimen was irradiated by the electron beam, the structure of the frozen water fundamentally changed.
Instead of contracting as expected, the amorphous frozen water actually expanded and eventually buckled in each small hole, explaining the increase in distance between particles at 13K compared to 81K.
The group solved the problem by reducing the diameter of the holes in the support grids to just 100nm – about 1,000 times smaller than the width of a human hair. Using these in tandem with a 300nm diameter electron beam eliminated the movement of the specimen. The smaller holes prevented any movement of the frozen water, and the decreased beam diameter meant a minimal area around each hole was irradiated, preventing movement of the grid.
They tested the set-up by determining six structures at 13K, and then replicated the studies with the original cryo-EM setup at 81K.
They found every frame captured at 13K showed less radiation damage and a better signal-to-noise ratio, showing more structural information can be gathered using liquid helium.
“More work is needed to see if even lower temperatures may help,” said Chris, “The lowest we could do with our resurrected old microscope was 13K, but the microscope companies are already developing new helium cooled stages to take advantage of this potential benefit.”
The work opens up a new frontier for cryo-EM – the potential to solve structures of far smaller proteins and molecules. It also raises important questions about how cryo-EM equipment for lower temperatures is developed. While the 100nm holed grids are key, large macromolecules and proteins that aggregate at the edge of holes are not well suited to them.
This – and the need for minimal irradiation of the water and the grid – will be vital when considering how to develop new cryo-microscopes and specimen supports that are optimised for liquid helium temperatures.
The worked was funded by UKRI MRC, Astex Pharmaceuticals and the Herchel Smith fund.
