Cryo-EM: How to Test the Waters with a Proof-of-Concept Study

How do you know if Cryo-EM is Right for You?

In-depth analyses of protein structure can be achieved using cryo-electron microscopy (cryo-EM), a powerful technique making strides in the field of structure-based drug design. The path to adopting cryo-EM in your drug discovery program, however, is not always straightforward. Initially, you may need to invest heavily in both infrastructure and training and/or recruitment efforts to ensure you have the facilities and staff needed to maximize the benefit of cryo-EM. It’s a big leap, and one that can bring payoff for structure-based drug design. How can you convince yourself (and other key decision-makers) that you are ready? 

One low-risk approach is to use a proof-of-concept (POC) study to test and justify the addition of cryo-EM to your research platform. Its purpose is to serve as an initial step that provides you with the highest chance of succeeding in future cryo-EM experiments, providing an opportunity to adjust and build the necessary protocols and resources. POC studies can also be used to explore the many stages and factors of cryo-EM sample preparation, a notoriously iterative yet important process.  

Choosing the right protein target for your proof-of-concept study

In an ideal world, a POC study would be focused on a target of internal interest that would ensure a quick win, and one that is also likely to be successful with cryo electron microscopy. With this approach, the benefit could be two-fold: you could familiarize yourself with cryo-EM processes and generate highly desirable data along the way. 

However, if your first choice of target is particularly challenging for cryo-EM analysis, it might not be ideally suited to a POC study. Many highly desirable drug targets lack obvious starting places for both protein engineering and cryo-EM methodology and may require additional stabilization approaches during sample preparation. Alternatively, they may require intensive data processing and 3D reconstruction after data acquisition, which can create a major strain on your budget and compromise the feasibility of implementing a POC study. In this situation, you might decide to select an alternative target candidate that is more likely to be successful with cryo-EM without the need for additional sample manipulation. If you are focused purely on a specific target or target class, you might need to bypass the pilot study step and dive straight into a full cryo-EM project. 

The most suitable target candidates for a cryo-EM POC study are often:

• Of interest to the discovery pipeline
• Monodisperse, active, and stable proteins
• Larger than 150 kDa
• Proteins with structurally characterized domains

The goal of a POC study is to provide a learning opportunity for you and your team, as you explore everything from protein biology to computational biology.  You can improve your chance of success by selecting a well-studied protein with sufficient supporting literature, and if possible, one with a documented crystal form – even if it is of lower resolution. Selecting a target that allows you to focus on the logistics of the workflow and to optimize the technique can set you up for a higher chance of success and provide the necessary foundation to broaden the scope of follow-on cryo-EM efforts to more challenging targets.

Navigating upstream workflow optimization and challenging targets

Once you have selected a target for a cryo-EM POC study, you will first need to optimize upstream stages of the workflow, such as protein purification and vitrification. In cryo-EM, the goal of sample preparation is to preserve the protein closest to its native state, in a way that provides maximum contrast for measurements and leads to the highest possible resolution. As sample preparation is a key determinant of success, exploring ways to optimize the process is critical. If your target protein of choice does not possess the qualities listed above, your POC study might involve more work. In this case, it may be helpful to seek advice from cryo-EM specialists who can elaborate on the following situations:

What if my target protein exhibits multiple conformational states?

Additional data acquisition and processing will be necessary to fully capture all the information. Alternatively, strategies to trap the protein in a limited number of conformations may be employed.

I have selected a target in the sub-100 kDa range. What can I do to get better results?

Adding mass via cognate binding partners or high affinity Fabs in a conformationally constrained manner can be highly beneficial. Note, however, that the affinity of these binding partners will need to be considered if negative stain is implemented as a QC check. Sometimes going directly to vitrification is warranted.

Should I add cognate binding partners to create a larger complex?

Proteins may require stabilization through binding partners that will increase the ordered mass of the target and provide more insight into the structure-function relationship of these dynamic regions.

Overcoming challenges of cryo-EM sample preparation

The clearest cryo-EM results are usually derived from highly pure, stable samples that show minimal compositional or conformational heterogeneity. Maintaining these characteristics during sample preparation can be challenging, as there are many opportunities along the way where protein quality can be compromised. Recombinant expression systems used to generate proteins need to be tested and optimized, to ensure they mimic the native cellular environment as closely as possible. The resulting protein must then be isolated through numerous purification and characterization steps and be kept amenable to vitrification prior to cryo-EM analysis.

One of the biggest challenges of cryo-EM sample preparation is identifying how to minimize potential air-water interface effects, a process that occurs when a protein adsorbs to the air-water interface in unsupported films of aqueous solution, often resulting in partial or complete denaturation. Air-water interface effects can be managed by identifying and implementing best practices related to blotting time and humidity, and using advanced sample preparation platforms.

To maximize the full potential of cryo-EM for examining complex dynamic proteins, you will need to make many decisions around your expression system of choice and the quality control checkpoints you will use at each step. Constructs will need to be engineered towards maximizing expression, stability and activity, and purification should be optimized for monodispersity, activity and stability. Furthermore, optimizing each of the factors that influence protein health must be balanced with other parameters. In early preparation efforts, for example, when little to no glycerol is added to the initial sample, the signal-to-noise ratio can be poor. In this instance, it helps to maximize protein concentrations where possible. However, it is better to start with a “happy” protein at a lower concentration than attempt to push a precious sample beyond its structural integrity.

Justifying joining the cryo-EM revolution

Cryo-EM is a powerful technique that allows you to explore the structures of proteins that have, so far, not been amenable to other structural approaches. When considering whether to adopt cryo-EM for the first time, however, it is not uncommon to encounter hesitation among team members or key decision makers. A POC study can be the perfect way to test and justify the addition of cryo-EM to your research platform and provides an opportunity for you to explore sample preparation protocols and resources needed to set you up for future cryo-EM success. 

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