Cryo-EM Provides the Structural Blueprint for Protein Nanomachines

At the molecular level, an organism's physiological function is largely dictated by the proteins it expresses, and in most cases of disease, a malfunctioning protein can be identified as the root cause. 

As with any "broken" or malfunctioning object, to find the source of the problem and to fix it, we must understand how it is built and the individual components that contribute to its function. Cryogenic-electron microscopy (cryo-EM) can lend a hand here, helping us to decipher the structural biology of important cell machinery such as proteins.

We spoke to Cristina Paulino, Assistant Professor in high-resolution cryo-EM at the University of Groningen, to learn about the role cryo-EM plays in her group's work in the study of membrane protein structure and function.

Molly Campbell (MC): Why is it important to know the structure and understand the mechanisms of membrane transporters? What specific types of membrane transporters do you typically study and why?  

Cristina Paulino (CP): Obtaining structural insights is fundamental to unravel the mechanism of action of proteins. When combined with functional and physiological data, we are able to understand in detail how these proteins operate, why and how they malfunction, what is the cause of a disease and how we can tackle it. I like to compare a protein structure to a blue print of an assembled machine. Whilst genetics and biochemistry help in understanding what the physiological role of a protein is, structural biology uncovers what these nanomachines look like and how they are wired. Only then can we rationally understand them and use this knowledge to repair, engineer or block them, for example, by building drugs that only fit this specific machine (structure-based drug design). 

My research is focused on elucidating the mechanism of action of proteins that are embedded in the membrane, specifically that of membrane transporters and channels. Although of high pharmacological relevance (> 60% of current drugs on the market target membrane proteins), the structure-function-relationship of membrane proteins is poorly understood and requires an interdisciplinary approach at the interface of biology, chemistry and physics.

While my group is primarily driven by the fundamental question on how these proteins work, results can have an additional social-economical application. This is demonstrated in our studies of the human ASCT2, the main source of glutamine uptake in human cells, which is strongly linked to cancer cell growth, poor patient survival and a new hot-target in cancer therapy. In addition to our work on unraveling the details of its transport mechanism, we collaborate with medicinal chemists to identify new inhibitors and guide structure-based drug design. Other studies blur the conventional conceptual boundaries present in classical transport mechanisms. The KdpFBAC complex, an emergency potassium uptake system in bacteria, for example, combines features of a primary-active transporter with that of channels. In the TMEM16 family, members can either function as calcium-activated chloride channels or lipid scramblases, or even both. Our studies demonstrate how in the course of evolution conserved protein architectures not only evolved from one another but can merge together to adapt to different environmental and cellular requirements.

MC: How integral is cryo-EM to your work? What difference does it make to the capabilities of your team?

CP: Over the past years cryo-EM has proven to be an indispensable technique in structural biology providing several advantages over X-ray crystallography, namely (i) requires only small amounts of protein; (ii) is not limited by the formation of protein crystals; (iii) is hardly limited by buffer composition and allows the induction of conformational changes prior to freezing; (iv) is not hampered by compositional or conformational heterogeneity of the sample, providing a glimpse into structural dynamics; (v) enables the determination of both low and high-resolution structures; and (vi) permits the use of tools that mimic a native lipid environment, like nanodiscs. Notably, while the resolution obtained with cryo-EM is on average lower than with X-ray crystallography, the common resolution obtained for membrane proteins is similar (about 3-4 Å). These advantages have proven to be crucial to tackle several challenges faced when working with membrane proteins, allowing unprecedented research and making cryo-EM often the technique of choice to study the structure of membrane proteins. As such, cryo-EM is the main technique employed in my group.

MC: What is the typical time frame for deducing the structure of a membrane protein using cryo-EM?

CP: In a perfect world with a perfect sample, access to a well-aligned high-end microscope and a powerful image processing cluster, one can solve a high-resolution structure within days. However, in reality, particularly when working with challenging membrane proteins, the time-frame is often weeks and months. 

MC: You recently used cryo-EM to determine the structure of the TMEM16 protein family. In a press release, you state that this approach “allowed you to sample the dynamics of the active structure”. Can you please expand on this? Why was cryo-EM superior to other methods available?

CP: One of the beauties of cryo-EM is that the sample is shock-frozen, retaining all conformations present in solution. This is in strong contrast to X-ray crystallography, where by definition all proteins in a crystal are in an identical state, which is often restricted to an energetically favored conformation. Also, we can cope with conformational heterogeneity present in cryo-EM images on the level of image processing. Thus, one can determine several structures, representing different functional states, of a protein in solution from the same sample. In the case of our recent work on the lipid scramblase TMEM16, we were able to obtain several structures of the scramblase under activating conditions. We interpreted these distinct conformations as snapshots of a stepwise activation mechanism of the protein, and propose that one of the intermediate states might be responsible for the non-selective ion-conduction observed in some TMEM16 proteins.  

MC: Are there any limitations to using cryo-EM in your area of research? If so, how can these limitations be overcome?

CP: It has been extremely exciting to witness how much and how fast cryo-EM has evolved over the past years. While the technique is continuously redefining its limitations and becoming more user-friendly, we still face several challenges. Whereas for X-ray crystallography access to fully-operational and maintained synchrotron beam lines is largely available and free, the overall costs in cryo-EM and the level of expertise required to operate the microscopes has been an obstacle. This has been, to some extent but not entirely, addressed by the implementation of government-subsidised cryo-EM facilities.

In my group, we have an in-house high-end microscope at our disposal, and we make sure to thoroughly train all group members. Other obstacles comprise the access to and the costs of image processing clusters, data management (several TBs of data can be recorded per day) and expertise in image processing. Luckily, IT costs drop with time and the implementation of graphics processing unit-driven software keep the costs affordable. Certainly, sample preparation has a lot of room for improvement (e.g. even less amount of protein required and avoid air-water-interface) with several groups and companies working on this problem.

Finally, the overall time required to solve a structure, and the limitation in size and the average resolution, still impose a challenge and hamper high-throughput approaches that are required in structure-based drug design. These problems are being addressed by a combination of more stable electron microscopes, better and more efficient electron detection cameras and improved processing algorithms.  

Cristina Paulino was speaking to Molly Campbell, Science Writer, Technology Networks.