Cryo-EM advancements lead to ‘resolution revolution’: Thermo Fisher
Thermo Fisher Scientific has added the E-CFEG cold field emission gun to its Krios Cryo-TEM (transmission electron microscope). The technology is designed to work together as part of four total components to offer researchers high resolution in single-particle analysis.
Cryogenic electron microscopy (cryo-EM) enables researchers to analyze 3D protein structures not readily crystallized for X-ray diffraction or used in nuclear magnetic resonance. Advancements such as the cold field emission gun reportedly make it possible for laboratory professionals to obtain atomic resolution not possible with other technologies.
Outsourcing-Pharma spoke with Stephen Brohawn, assistant professor at the University of California-Berkeley's Helen Wills Neuroscience Institute, about how technological advancements, such as the newly launched features in Thermo Fisher’s cryo-EM solutions, have aided work in his lab, such as important work related to the COVID-19 virus.
OSP: In general, how has evolution in lab technology enabled you and your team to achieve more in your work?
SB: There have been tremendous advances in detector hardware, with direct electron detectors that allow for imaging at higher levels signal to noise. Also, maybe more importantly, they allow us to correct for beam-induced particle motion.
Microscope hardware is another improvement, with brighter, more coherent sources that produce a more stable beam, in addition to critical improvements to the software, because cryo-EM reconstructions are very computationally demanding and difficult.
OSP: Specifically, how have cryo-EM advances elevated your research?
SB: A few years ago, we would have never thought that these kinds of projects would be possible. Now, we’re able to see small membrane proteins and very flexible proteins at high resolution.
We can analyze more difficult samples with single-particle analysis. We can even think about doing drug discovery with cryo-EM, where you need high-resolution structures to interpret the chemical interactions between small molecule drugs and proteins.
OSP: Please tell us a bit about your TASK-2/SARS-CoV-2 3a protein work.
SB: As we recently shared with Thermo Fisher Scientific, TASK-2 is important for a number of physiological processes. One is its role in regulating breathing rate in response to changing CO2 levels in the blood.CO2 impacts solution pH and TASK-2 is a pH-regulated potassium channel. We solved cryo-EM structures of TASK-2 at different pH values to understand how these changes in pH open and close the channel, down to the atomic-scale rearrangements that make it happen. What we saw is that, compared to other ion channels, protons inhibit TASK-2 in two totally new ways.
We tried doing this with crystallography previously because TASK-2 is a very small membrane protein (~65 kDa), which puts it at the low end of what is feasible for cryo-EM. Even a few years ago, I would have said that it’s probably not going to be possible to analyze proteins this small any time soon.
However, a very talented postdoc Dr. Baobin Li decided to give it a shot, and we worked out ways to use lipid nanodiscs to determine TASK-2 structures at ~3.5 Å resolution. Without cryo-EM, we wouldn’t have been able to solve these structures and work out the mechanistic underpinnings of how this channel is regulated.
Using new hardware in collaboration with Abhay Kotecha at Thermo Fisher Scientific, we have improved the resolution to 2.5 Å resolution.
Right at the beginning of the pandemic, when it became clear that this was a new virus, we started to look at its genome. From the literature on other coronaviruses, we saw that there were at least two or three putative ion channels that the virus encodes. We studied these channels to potentially contribute to the knowledge base for the virus, helping future vaccine or therapeutic development.
One of these channels is called ORF3a, or just 3a, and it has been shown that deleting it from the related SARS-CoV-1 virus reduces viral maturation and morbidity in animal models. We thought that this looked like a promising and understudied target, so we investigated 3a in two ways, studying both its function and structure.
In a few short weeks of very intensive work, another very talented postdoc in the lab, Dr. David Kern, worked out a way to purify this 3a protein from SARS-CoV-2, reconstituted it in nanodiscs, and determined its structure. We were able to generate a fairly high-resolution reconstruction on our equipment here at Berkeley (2.9 Å) but were excited to collaborate with Dr. Abhay Kotecha from Thermo Fisher Scientific to see if we could push this resolution even further.
We took our extra grids, shipped them to the Netherlands, and crossed our fingers. Using the data collected at Thermo Fisher, we were able to generate reconstructions to 2.1 Å resolution, which is really quite remarkable, especially given the small size of this target (62 kDa).
Outsourcing-Pharma also spoke with Kotecha, senior applications development scientist at Thermo Fisher, about the advancements in cryo-EM technology, notable projects he has observed, and what such changes might mean to researchers in life sciences and drug development.
OSP: When evolving the tech in cryo-electron microscopy, what feedback have you heard from customers (especially in life-sciences research and drug development) that informed the innovations?
AK: When I used the Krios G4 cryo-TEM, I was surprised and really couldn’t believe the images I was seeing. I was looking at GABAA receptor, a small human membrane protein.
This protein was difficult to observe because there was hardly any contrast and required either Volta phase plate or high defocus to be able to see these particles, both these methods are detrimental to high-resolution information, but with the Selectris X energy filter and Falcon 4 detector, I could clearly see the particles of this receptor with high contrast at very close to focus.
This project was a collaboration with Prof. Radu Aricescu at the MRC Laboratory of Molecular Biology, Cambridge. Prof. Aricescu is working on structure-based drug design for neurological disorders. For drug development, accurate structures with atomic details and water molecules are very important. Such information can be obtained at a resolution of 2.5 Å or higher.
Because of high contrast and, hence, better signal with our new technology, Radu’s team was able to resolve multiple structures of GABAA receptor bound to several different clinical drug targets resolving to 2.2 Å and higher. Two of the structures were resolved beyond 2 Å. With this new hardware and improvements in data processing, we are seeing similar improvements in resolution across multiple soluble and membrane proteins.
OSP: Please feel free to go over the new/notable features and tell us how they help researchers like Dr. Brohawn.
AK: The last seven years have seen a remarkable technological evolution in electron cryo-microscopy and it has started a ‘Resolution Revolution’ in the field. This has opened a whole new era in structural biology. However, the attainable resolution for many protein structures was limited to 3 - 4 Å resolution range.
With the introduction of new hardware that we proudly call ‘power of four’: Krios G4, E-CFEG, Selectris X energy filter, and Falcon 4 detector, in addition to advanced developments with the data processing software, we have taken this revolution to the next level.
For the first time last year, this technology made it possible to reveal mechanistic details of a standard protein sample at a true atomic resolution going to 1.2 Å resolution and allowing us to see each individual atom in the cryo-EM map. These technological advances also allowed us to observe the structure of membrane protein and a major drug target, a human GABAA receptor, to 1.7 Å resolution for the very first time. Finally, we were able to see a network of hydrogen atoms that were never seen before on membrane proteins.
At the peak of the pandemic, I came across fantastic work from Prof. Brohawn’s lab on a very small membrane protein from SARS-CoV-2, a 62kDa 3a ion channel reconstructed to 2.9 Å. Such sub-100 kDa proteins are extremely challenging to resolve at high resolution since there is a very small mass to align and noise dominates the signal.
When we collected data of this sample using Krios G4 (equipped with E-CFEG, Selectris X, and Falcon 4), tiny particles of this ion channel were easily visible and with very high contrast. This allowed Brohawn’s group to generate reconstruction of this protein to 2.1 Å resolution.
At this resolution, they can accurately build the atomic model and clearly see a network of water molecules. Such a detailed cryo-EM map allowed them to understand the function of this protein as an ion channel with a novel fold. This work is important because this ion channel could represent a new target for drug discovery for treating COVID-19.
I have worked on several challenging protein samples. In every case, the resolution was significantly improved revealing important structure details. With such advancements, in hardware and software, I feel strongly that we are seeing a second resolution revolution in the field.
