The future is frozen

Cryo-electron microscopy (cryo-EM) may not yet have revolutionised the world of medicine but it has definitely transformed the field of structural biology.

In October, Scottish physicist turned molecular biologist Professor Richard Henderson from MRC Laboratory of Molecular Biology, Cambridge, UK, along with Jacques Dubochet, University of Lausanne, Switzerland, and Joachim Frank, Columbia University, New York, was awarded the 2017 Nobel Prize for Chemistry “for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution”.

The technique allows researchers to freeze biomolecules mid-movement and visualise processes they have never previously seen, which is decisive for both the basic understanding of life’s chemistry and for the development of pharmaceuticals, according to The Royal Swedish Academy of Sciences. Their technology has moved biochemistry into a new era, according to the academy.

Professor Henderson will soon be visiting Australia to present at the 43rd Lorne Conference on Protein Structure and Function, to be held from 4–8 February in Lorne, Victoria. Henderson will provide an overview of the current state of cryo-EM, and discuss “how there are still many improvements that can be made before the approach reaches its theoretical limits”.

Making a near-perfect method

Recent years have seen major technology advances in cryo-EM but there is still a long way to go. “Although the field of single particle electron cryomicroscopy (cryoEM) is already producing many valuable structures that cannot be obtained by any other method, there are still a number of significant improvements that we expect to make the method even more productive,” said Henderson.

“We expect the main developments to be (a) bigger, faster and better electron imaging detectors, (b) more robust and reliable quarter phase plates and (c) a reduction in beam-induced specimen motion which blurs the images, obtained by improvements in the specimen supports,” he said.

“Our goal is to make cryo-EM into a near-perfect method limited only by fundamental physics, such as radiation damage, which is the only fundamental limitation,” said Henderson.

Over the last 20 years, the great improvement of the power of conventional synchrotron X-ray beam lines has been one of the most exciting developments in structural biology, Henderson said. “These X-ray sources have been made brighter and more automated and have been equipped with more efficient X-ray counting detectors, so that macromolecular crystallography is now producing over 10,000 new structures deposited in the Worldwide Protein Data Bank (PDB) each year, from a wider range of specimens using smaller and smaller crystals. There are now 135,000 macromolecular coordinate datasets deposited and downloadable by anyone who wants to use them from the PDB.”

Molecular medicine

In the past few years, cryo-EM has been used in a variety of scientific studies, from Zika virus to antibiotic resistance. Biochemistry is now facing an explosive development and is all set for an exciting future, according to the academy.

When asked about how cryo-EM could transform the future of health and medicine, Henderson said, “I’m afraid cryoEM has not (yet) transformed health and medicine. It is still too early and the method has only been operating to produce really high-resolution structures since around 2013.”

X-ray crystallography took 30 years to become useful for “structure-based drug design” — from 1959 when we had the first (myoglobin) protein structure until 1990 when it was adopted by many pharmaceutical companies, said Henderson.

“CryoEM will not take so long, because numerous pharma companies have already started to use it. In the slightly longer term, cryoEM will be used to help to develop many new drugs — drugs that either activate or inhibit a particular disease-related process — to either cure or ameliorate the problem.”

The cost challenge

Cost, however, is one of the major factors limiting the spread of cryo-EM. “We are hoping to make cryo-EM less expensive — to democratise it,” said Henderson.

In a paper published in Cambridge University Press Journal Quarterly Review of Biophysics, Henderson and his colleague Kutti R Vinothkumar discuss the need for affordable cryo-EM. “At present, the performance advantage of ‘high-end’ cryoEM (‘high’ because of the large associated capital and running costs of a 300 keV facility) means that those groups and institutions that have access to the best equipment have an enormous advantage over those without such access,” the authors said in the paper.

“Since an installation with equipment that can deliver the best quality images can cost £5m (AU$8.7m) with annual running cost including management in the range of ∼£250,000 (AU$433,516), this acts a barrier to providing access for research groups that are not located in a major centre. One solution to the problem would be to provide national (eg, Saibil et al. (2015) for cryoEM in the UK) or international facilities best illustrated by the success and wide availability of third generation synchrotron sources for X-ray diffraction studies (eg, ESRF, SPring-8).

“The preparation of suitable specimens for cryoEM also requires a lot of preliminary evaluation. Alongside the need for excellent biochemistry, there are many pitfalls along the route to producing a perfect cryoEM grid with a good distribution of single particles that are not denatured at the air–water interface, aggregated, stuck to the support film or suffering from preferential orientations.

“To overcome this list of typical problems requires (preferably) daily access to a cryoEM facility that is good enough for characterisation of any specimen preparation problems, and for collection of small diagnostic datasets. High electron energy is not necessary in such a diagnostic tool since good images can be obtained at 100 keV. However, the coherence of the electron source makes an enormous difference to the detail visible in the highly defocussed images that are needed to observe internal structure in smaller proteins. At present, it is not possible to interpret clearly images of protein assemblies of 150 k Da without the higher defocus that can be used with the much higher coherence of a field emission gun (FEG).”

Diagnostic cryoEM

Thus alongside the availability of state-of-the-art ‘high-end’ electron cryomicroscopes, the structural biology field also desperately needs an inexpensive diagnostic cryoEM, the authors said in the paper. “Such an instrument is needed for preliminary evaluations, and should be able to achieve good enough resolution to evaluate the intrinsic quality of the specimens once a suitable particle distribution has been obtained.

“This local characterisation of specimens and grids could then feed into and make the best use of regional, national or international resources where higher resolution cryo-microscopes with greater automation could be available.

“It is certainly unrealistic to expect every laboratory to be able to afford a state-of-the-art facility, which at present needs to include a 300 keV Krios or similar high-end instrument, plus a direct electron detector and possibly also a zero-loss energy filter.

“Given the cost of these higher voltage microscopes (due to the need for X-ray shielding and high voltage power supplies), it would be sensible to aim for a 100 or 120 keV instrument for the general market with a FEG electron source (500x brighter than a tungsten filament) and an efficient inexpensive detector at perhaps one tenth of the cost of the ‘high-end’ instruments.”

Image caption: Ground-state bacteriorhodopsin structure (purple) compared with a triple mutant (yellow) that resembles the trapped M-intermediate, from Subramaniam & Henderson, 2000. Image courtesy of MRC Laboratory of Molecular Biology.