This study is all about the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA are based on data acquired on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment, although this comes with performance limitations. Here, a multi-purpose TEM with a side entry cryo-holder was used to evaluate the potential of high-resolution SPA, resulting in a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. This map clearly showed two positions of an aromatic side chain. Further, examination of optimal imaging conditions depending on two different multi-purpose electron microscope and camera combinations was carried out, demonstrating that higher magnifications are not always necessary or desirable.
Fig: 3 Å (global estimated resolution) cryo-EM map of mouse heavy-chain apoferritin. (A) Map coloured by local resolution; one quarter sliced away to allow visualisation of internal density. Scale bar 2 nm. (B) Representative helix (Leu48-Arg77) from one subunit, with PDB: 2CIH fitted. Map is contoured at 3σ. Black arrows show two rotamers of Arg63. (C) Nine representative sidechains. Red arrow shows a metal density.
Since automation is effectively a requirement for large-scale data collection, and augmenting the multi-purpose equipment is possible, we expanded testing by acquiring data with SerialEM using a β-galactosidase test sample. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.
Kayama, Y., Burton-Smith, R.N., Song, C. et al. Below 3 Å structure of apoferritin using a multipurpose TEM with a side entry cryoholder. Sci Rep 11, 8395 (2021). https://doi.org/10.1038/s41598-021-87183-1