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Cryo-EM Opens New Avenues in Channel and Membrane Protein Research

By 05.06.2021

Membrane protein not only dictate how our cells communicate and function in unison but are also the key that viruses and bacteria use to gain entry to our cells. This class of protein, however, has been a challenging target for traditional structural analysis, requiring highly precise sample preparation or being entirely incompatible with crystallographic analysis.

Using cryo-electron microscopy (cryo-EM), and what we call “the power of four,” Dr. Stephen Brohawn and his team at the UC Berkeley Department of Molecular and Cell Biology were able to push the bounds of membrane protein research and solve critical structures, including the TASK-2 potassium channel and SARS-CoV-2 3a protein. Dr. Brohawn joined us to talk about his research for the launch of the Thermo Scientific E-CFEG, a new cold field emission gun and value-added component for the Thermo Scientific Krios Cryo-TEM.

Thank you for joining us! Can you briefly introduce your lab and your work?

My lab studies the molecular basis for sensory transduction and electrical signaling in the nervous system. We are very interested in figuring out how ion channels in neurons that generate electrical signals work at a molecular level.

Why are channel and membrane protein a focus for cryo-EM research?

Crystallography works for membrane proteins, but it’s very challenging. One difficulty is that you need some way to shield their hydrophobic portions that are usually surrounded by the lipid membrane in the cell.  It often takes quite a lot of protein engineering and various kinds of tricks to get these membrane-protein crystals to form.

Besides the fact that it’s difficult, we would also prefer to look at membrane-protein structures in conditions that are closer to their native cellular environment. This makes cryo-EM very appealing because we can reconstitute membrane proteins into things like lipid nanodiscs that mimic the cell membrane much more closely than a detergent micelle ever could.

Can you tell us a bit about your recent work on the TASK-2 potassium channel?

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 wanted to use a structural approach to understand how these changes in pH open and close the channel, down to the atomic-scale rearrangements that make it happen.

To do this, we solved cryo-EM structures of TASK-2 at different pH values, and 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 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.

What are the advances in cryo-EM that have enabled your current research?

I think it’s a combination of factors that evolved and converged at the same time.

For one, there have been tremendous advances in detector hardware, with direct electron detectors that allow for imaging at higher levels of 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 software, because cryo-EM reconstructions are very computationally demanding and difficult.

Can you tell us about your work on the SARS-CoV-2 membrane protein?

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 wanted to study 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, one of the postdocs in the lab, 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 when Abhay Kotecha from Thermo Fisher Scientific asked to collaborate with us 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).

Cryo-EM structure of dimeric 3a ion channel from SARS CoV-2 membrane protein research.

Cryo-EM structure of dimeric 3a in lipid nanodiscs at 2.1 Å nominal resolution viewed from the membrane plane.

Broadly speaking, our collaboration with Thermo Fisher has improved the resolution of both structures by about 1 Å, and allowed us to see things that we weren’t able to in our previously published lower-resolution maps. For both the TASK-2 and SARS-CoV-2 projects, we used the new instrumentation from Thermo Fisher that consists of the Falcon 4 Camera, the Selectris X Imaging Filter, and the E-CFEG.

You can imagine that all the components are playing a part, for instance the energy filter clearly improves the contrast of the images by allowing only certain electron energies through to the camera.

Older XFEGs were optimized for brightness and they run at high temperatures, whereas the CFEG is optimized for narrower energy spread, leading to less blur. For high-resolution work, this can be really important. The software that comes with the CFEG also alleviates some of the concerns for cold sources, like the accumulation of gaseous atoms on the tip that need to be regularly burned off.

We’re excited to keep pushing the envelope here and to make careful comparisons to see what we can really do with this combination of CFEG, filter, and camera and why we see the improvements we have.

Seeing the scientific community come together and collaborate like this is admirable.

It’s something that we are real strong believers in, and this is a great example of how open source and pre-printing can really accelerate science.

We were only able to initiate this collaboration with Abhay because we posted a pre-print of our first structure in June of last year, just a couple months after starting the project. If we had waited through the traditional publication timeline, it would have taken several months and who knows where we would be.

What do you see in the future of cryo-EM?

The field is developing so fast that it’s very exciting to be a part of it. 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.

Dr. Stephen Brohawn is an Assistant Professor at UC Berkeley Department of Molecular and Cell Biology and the Helen Wills Neuroscience Institute.

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Alex Ilitchev is a Science Writer at Thermo Fisher Scientific. This interview has been edited for length and clarity.

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