Cryo-electron microscopy, commonly known as cryo-EM, has emerged as a powerful technique in the field of structure-based drug design. With cryo-EM, you can explore the structures of large protein complexes or membrane proteins that have, so far, not been amenable to the traditional structural techniques.
Incorporating cryo-EM into your workflow can generate many benefits by providing conformational and mechanistic insights into proteins in near-native states. Despite the advantages that cryo-EM offers, many scientists consider the technology to be out of reach.
When you’re thinking about adopting a new workflow or technology like cryo-EM, there are always challenges and barriers that must be overcome. Here we share some challenges you might face when adopting cryo-EM, and tips for how to overcome them.
Finding solutions for the impossible with Cryo-EM
Until recent years, the field of structural biology has been powered by X-ray crystallography and nuclear magnetic resonance (NMR). Both methods have generated invaluable information for the drug discovery community, and collectively have produced over 130,000 open-access structures. Despite the capabilities of crystallography and NMR, their limitations have meant the structures of many high-value drug targets of interest remain out of reach.
Large protein complexes and membrane proteins are among those target groups which are difficult or impossible to characterize with traditional structural techniques – and can be captured with cryo-EM. Thanks to the flash-freezing method used in cryo-EM, you can examine multiple protein conformations within the same sample. Such capabilities create a world of possibilities, enabling you to:
- Design drugs that target specific conformational states
- Study underlying mechanisms of action that are unattainable by other means
- Identify drug binding sites and apply greater selectivity
- Structurally enable targets previously considered intractable due to challenges with crystallization and/or size
- Dive into medicinal chemistry efforts in structure-based drug design, with unprecedented details of drug-protein interactions to guide compound design
But what are the challenges that must be overcome to adopt cryo-EM in your program?
Challenge 1: Sample preparation is an iterative procedure
Sample preparation for cryo-EM can be overwhelming, as it is a highly involved process requiring a significant investment of time. Before capturing images with a transmission electron microscope, proteins in the sample must be purified and isolated, prior to vitrification (a flash-freezing method). Vitrification is a critical process which allows you to capture near-native protein states; however, it can also place significant stress on your protein of interest.
Air-water interface effects can render a seemingly high-quality protein sample unsuitable for imaging. Potential detrimental effects include protein aggregation, denaturation, complex dissociation and orientation issues, each of which must be ruled out during sample preparation through the use of strategic checkpoints.
Throughout the process of sample preparation, you will find there are many variables, including sample characteristics, vitrification methods, additives, and sample supports – each of which require extensive exploration, optimization and continuous problem-solving. Establishing reproducible conditions is particularly important in the setting of drug discovery, to reduce time to and cost of subsequent structures. Overall, there are no shortcuts. Regardless of the effort required, efficient and successful target enablement efforts are possible when you adopt well-tested troubleshooting workflows in combination with next-generation sample preparation methods.
Challenge 2: You need to upskill your team
Due to the complex nature of cryo-EM, it is important to have experienced staff at hand during the troubleshooting process and throughout operation. Without experience on your side, navigating the multiple variables at play would be a highly inefficient and frustrating experience. As cryo-EM is a rapidly emerging field, you may not have had the opportunity to work on cryo-EM projects during your career as a structural biologist and could need additional support.
Structural biologists without prior cryo-EM experience will typically undergo rigorous training to expand their skillset. Given the demands of other ongoing research projects, you might be wondering how you could possibly have time to learn enough about cryo-EM to incorporate it into your laboratory. It’s true: the entire workflow does require a diverse set of skills, including protein design, sample preparation, data processing, 3D reconstruction, model building, and more.
It might sound like we are trying to deter you from adopting cryo-EM, however this couldn’t be farther from the truth. Rather, we feel it’s important to acknowledge the steep learning curve and tackle it appropriately with immersive training sessions or additional recruitment. Many other structural biologists have made the leap from cryo-EM newbie to seasoned expert – and have done so by tactically absorbing the expertise of others.
Challenge 3: You need to invest heavily in infrastructure
There’s no denying it: cryo-EM bears a high price tag. To establish an in-house facility, your laboratory will need to invest heavily and cover many ongoing costs. To start with, you’ll need to acquire specialized laboratory space, a microscope, and set up ancillary equipment. After installation, you’ll need to schedule regular maintenance work, obtain high-end computational support and storage infrastructure, and invest in recruiting experienced microscopists and/or in-depth technical training for internal scientists.
Given cryo-EM is technically cumbersome and requires heavy investment, decision-makers are often reluctant to incorporate such a high-risk technique into research projects via internal facility investment. Ultimately, it comes down to the bottom line, and for many organizations starting with a proof-of-concept project makes sense as it allows them to engage in lower-risk programs to determine technical feasibility and develop internal workflows.
For structural biologists and drug discovery researchers, the unprecedented biological output potentially attainable using cryo-EM supersedes all the challenges that need to be overcome on the road to adopting it.
Joining the cryo-EM revolution
Cryo-EM is an involved process, but it is also undeniably a powerful technique that can take your projects to a whole new level. By exploring the practical considerations associated with expanding your structural biology toolkit, you can give yourself the best possible chance of propelling your drug discovery project forward.