The first step of cryo-EM is having an appropriate target!
One of the great things about cryo-EM is that it is a technique that can be used to determine the structure of many kinds of proteins and protein complexes. However, some proteins represent more challenging targets than others.
Cryo-EM is fundamentally an averaging technique, whereby multiples of low signal, high-noise images are averaged together to increase the signal-to-noise ratio. In order to do this, many images of the same protein conformation in the same view are necessary. Anything that decreases the similarity of the particles to one another increases the amount of particles you will need.
Factors affecting how straightforward the process can be are:
-Soluble vs. membrane proteins
While cryo-EM is a powerful tool for determining the structure of membrane proteins, soluble proteins are often an easier target, as they may require less optimization to get a good distribution of the protein on the grid. There are also a wide range of membrane mimietics available- not only may your protein of interest behave better in one than another, but they can all behave quite differently on the cryo-EM grid.
– Protein size
While proteins as small as ~50kDa have been determined by cryo-EM, these represent extremely challenging targets. In general, proteins above 200kDa are considered more promising targets. In order to obtain a structure, the particles must be aligned to each other accurately, and this can be difficulty to do with small proteins. Larger targets with more features are easier to identify on the grid, and easier to align during reconstruction.
-Flexibility
The good news is that cryo-EM can be used to solve not just a single structure, but also to pull out different conformations that may be present in the sample. However, if the protein is highly flexible, each particle will contribute to a single conformation, and thus more particles are required.
-Symmetry
The higher the order of symmetry (2-fold vs. 3-fold. etc) a protein has, the more ‘copies’ of the protein are essentially present in the same sample. This means that fewer particles are required to reach an equivalent resolution.
-Number of binding partners
Protein complexes may be harder to solve than single-chain proteins, as the complex can fall apart on the grid. The more subunits the complex has, the greater the number of different species the complex can dissociate into.
In practice, a combination of these factors will determine how challenging a project will be. More challenging projects will likely require a lengthy optimization process. Once you have identified a target for structure determination the first step is to assess the protein by negative stain EM.