Tips for purifying and analysing cryo-EM samples

Cryo electron microscopy (cryo-EM) has become a popular tool for determining the structures of macromolecular complexes at the atomic resolution. In this method, frozen hydrated specimens are imaged by electron microscopy at cryogenic temperatures. This means that the specimens remain in their native state without the need for dyes or fixing, enabling the visualisation at molecular resolution of fine cellular structures, viruses and protein complexes.

In structural biology, X-ray crystallography is well established to determine the three-dimensional structure of proteins; however, for challenging proteins, such as membrane proteins and larger protein complexes, this method has posed challenges. With the recent technical improvements, cryo-EM has become a very important and widely used technique within structural biology. The total number of structures determined using cryo-EM are increasing rapidly.

The cryo-EM workflow starts with cloning and expression of the target, which is then purified and characterised before the sample is applied on grids and analysed with cryo-EM (Fig 1).

Fig 1. The cryo-EM workflow: After cloning and expression, the target protein is purified and characterised. Finally, the sample is applied on grids and analysed with cryo-EM.

Good protein characterisation and protein purification are imperative to yield a sample with the quality required for successful cryo-EM.

To purify a protein of interest, the question always remains how many steps are needed and what methods should be used. A combined chromatography method to try would be the use of an affinity tag step followed by size exclusion chromatography. As with any tag purification, we suggest starting with a His-tag. If that does not work, try a Strep-tag™ II for better purity, or GST for improved solubility.

For the size exclusion chromatography (aka gel filtration), it is best to choose a resin based on the size of your target molecule. At Cytiva we offer:

• Superdex 200 Increase: M_r ~ 10 000 to ~ 600 000 for molecules such as antibodies
• Superose 6 Increase: M_r ~ 5000 to ~ 5 000 000 for large molecules such as protein complexes
• Superdex 75 Increase: M_r ~ 3000 to ~ 70 000 that can be used with most recombinant tagged proteins
• Superdex 30 Increase: M_r ~ 100 to ~ 7000 that is suitable for peptides and other small biomolecules.
   

To learn more about the protein preparation for cryo-EM, watch our recent webinar.