Leading the Charge in the Cryo-EM Resolution Revolution

Who could have believed 100 years ago, or even 50 for that matter, that today we would be able to study cells and biological systems with such depth and detail? This revolution has brought with it new understanding of the pathologies and mechanisms behind disease and opened doors to previously inconceivable diagnostics and treatments.

To continue this march of discovery and development, scientists must look beyond the cell to the structures on the surface and within. This is where structural biology comes in. Armed with techniques including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryogenic electron microscopy (cryo-EM), the structures of anything from cellular machinery to ion channels and receptors can be revealed and utilized. Cryo-EM in particular is set to play an increasingly important role in future studies, with recent advances in resolution that are enabling scientists to make observations at the atomic level with this technique.

We spoke to Steve Reyntjens, Director of Product Marketing for Cryo-EM at Thermo Fisher Scientific, about how cryo-EM advances are changing scientific research and the role that their precision instruments are playing in this.

Karen Steward (KS): Atomic resolution in cryo-EM is a relatively recent phenomenon, how do you see it is changing the research landscape?

Steve Reyntjens (SR): In recent years, tremendous progress has been realized in the achievable resolution for cryo-EM due to technological advances in detectors, automation of data collection on very stable microscope platforms and data processing. This has made cryo-EM a viable technique for structure-based drug discovery and has led to a broader adoption in structural biology. That transition was fueled by “the resolution revolution”, after a seminal Science paper by Werner Kuhlbrandt from 2014.

Now six years later, we’re at the dawn of a second resolution revolution. Earlier this year, using the newest technologies from Thermo Fisher, scientists have been able to break the atomic resolution barrier with cryo-EM. This breakthrough will allow scientists and researchers to study how proteins work in unprecedented detail, giving them the ability to resolve protein structure well below 2 angstroms, which reveals never-before-seen details. At this resolution, even the smallest improvements open up a whole new universe of information and we believe this new resolution revolution is going to fuel cryo-EM as the method of choice for both pharmaceutical and academic research.

As an example, scientists have been able to resolve the structure of a protein called GABA_A receptor to an unprecedented resolution of 1.7 angstroms, whereas the previous best structure was 2.5 angstroms. The GABA_A receptor protein is a human membrane protein and is a target for many drugs, such as anesthetics and anxiety medication. This new structure reveals how a small molecule – histamine – is displacing water molecules in a drug-binding pocket, an insight that could potentially lead to drugs with fewer side effects.

KS: What are the main problems caused by noise in TEM, and particularly atomic resolution cryo-EM imaging?

SR: This points to some of the most fundamental challenges of cryo-EM – and more particularly single particle analysis (SPA), the technique that is used to determine 3D structure of proteins. With SPA, we irradiate fragile biological molecules with highly energetic electrons. The damage that is caused by this electron bombardment is essentially altering the sample, and this damage accumulates throughout the exposure, essentially limiting the number of electrons you can use to image before the molecule is too damaged.

So, nature forces us to work with very low electron doses, resulting in correspondingly weak signals on the detector. Therefore, it is critical to reduce the noise in the detection system as much as possible, as this will directly impact the quality of the end result. Of course, the higher the targeted resolution, the bigger the signal-to-noise challenge, and thus to achieve atomic resolution cryo-EM, users need to maximally optimize every single element in the electron microscope and detection chain to get the most out of every electron.

KS: How do the Selectris X imaging filters overcome problems seen in imaging?

SR: The Selectris X imaging filters play an important role in separating useful image signal from unwanted noise and perform their function in a bigger setup of innovative technological components that make up a powerful cryo-EM.

The Krios G4 cryo-TEM provides a stable and robust cryo-EM platform, with state-of-the-art electron optics and sample manipulation capabilities. Combined with an electron source with low energy spread, this allows for the best possible delivery of the tolerable electron dose to the sample. On the detection side, the Falcon 4 provides an extremely efficient camera to convert the electron signal into images that can be processed on-the-fly or offline. Specifically, for the Selectris X filter, this is where the “noisy” electrons get separated from the electrons that carry to useful image signal, so that only the latter reach the Falcon 4 detector. What makes the Selectris filters unique is their extremely high stability, which allows precise filtering during the many hours that automated data collection happens.

This unique combination of filter stability, highly precise filtering, and exceptional optical performance are combined with the Krios G4 platform and Falcon 4 camera, to overcome the electron dose limitations and lead to atomic resolution cryo-EM.

KS: From a user perspective, how much of a difference is applying this technology likely to make to their workflows?

SR: Of course, as already discussed, the improvement in attainable resolution is going to enable cryo-EM to reveal more fundamental biological insights, which will impact drug discovery applications and allow us to better understand how drugs perform. However, these results will continue to depend strongly on sample quality, and atomic resolution will continue to be very challenging for many samples.

In addition to the resolution leap, the Selectris technology will also significantly impact productivity. By having a better filter and camera, scientists will be able to achieve a specific resolution with much less data. The exact gain is strongly dependent on the specifics of the experiment and sample, but in many cases, productivity will be boosted significantly.

And finally, all these advantages will be more easily attainable, thanks to the phenomenal stability, and tight integration of the Selectris filters.

KS: Can you tell us about some real-world examples where this technology is making a difference in research?

SR: While Selectris technology is still new, we are seeing great interest from academic researchers, as well as from customers in biopharma, to bring this technology into their cryo-EM workflows. At this time, the scientific results and full impact on research have yet to be determined, but together with researchers at the MRC Laboratory of Molecular Biology in Cambridge UK, we have been able to determine the structure of the GABA_A receptor, human membrane protein – now in combination with multiple different drug compounds. Using Selectris X the results are available fast and reveal more details than ever before. This can lead the way to optimize these drugs in terms of their potency and reduced side effects.

As another very promising example, we were recently able to support researchers at the UC Berkeley to resolve the SARS-CoV-2 3a ion channel at a much higher resolution than previously possible, using a Krios with Selectris X. The 3a ion channel is thought to be involved in viral release and inflammasome activation. It is also a potential druggable target, suggesting that therapeutics targeting 3a could potentially lead to treatment of a range of coronaviral diseases like SARS and COVID-19.

Steve Reyntjens was speaking to Dr Karen Steward, Science Writer for Technology Networks.