Recent Advances and Best Practices for Cryo-EM Analysis for Protein Structure Determination

Since the emergence of direct electron detectors and the improvement of software and hardware, cryo-electron microscopy (cryo-EM) has become a technique that routinely allows researchers to determine the structure of macromolecules to near-atomic detail^1,2. This acceleration has been catalyzed by an extremely fast-paced series of new developments, from sample preparation to data collection and processing. To allow for a wide democratization of this technique to non-experts, more efficient and accessible pipelines have been created. Strikingly, less than 10 years after the “resolution revolution”, cryo-EM has become an essential technique for protein characterization, both in academia and the industry^3,4.

One of the most remarkable aspects of the cryo-EM research community, has been the willingness to openly share protocols, strategies, and software⁵. This openness of technology and expertise contributed significantly to the very rapid pace of development, but some bottlenecks continue to prevent it from being fully accessible. In this method collection, we aim to describe some of the recent developments in the preparation of grids suitable for structural analysis by cryo-EM, which is, together with access to the data analysis hardware, the major bottleneck for many projects. We also discuss new protocols that are currently in development in this field, for new approaches in cryo-EM analyses.

As stated above, one of the current bottlenecks for the routine application of cryo-EM in the laboratory is sample preparation. Several issues can be encountered at the level of sample application to the grids: most commonly, the preferred orientation of the sample in ice, and its denaturation at the air-water interface. Shielding the sample from the air-water interface is one solution to this problem, by using support films of carbon or graphene that physically prevent the sample from being in contact with the air. To facilitate the application of thin films onto grids, de Martin Garrido et al.⁶ have developed a support floatation block produced by a 3D printer that allows small drops of water and sample to be applied in a controlled way. Using this block, the graphene and carbon layers are deposited in an inexpensive and user-friendly way.

Another way to prevent the damage at the air-water interface is to limit the time the protein is exposed to the atmosphere. For this, two different instruments, developed by Klebs et al.⁷ and Budell et al.⁸, use novel approaches for fast (as low as 10 ms) grid preparation, reducing the likelihood of issues with particle orientation. Another advantage of these devices is the possibility of performing time-resolved experiments, an exciting new frontier for structural studies.

In a contrasting approach, Nguyen et al.⁹ report a method to freeze grids for cryo-EM analysis in the absence of commercial or bespoke devices, which are either very costly or difficult to build. They show how grids of similar quality can be obtained using a cheap, home-made device that does not require any expensive instrumentation, contributing to the democratization of cryo-EM.

In cryo-EM, the sample of interest is not always a protein, but can instead be a small molecule or a large biological system such as a whole cell. The preparation of such samples is unique, and it requires specific steps, as well as different strategies in data collection.

For microcrystal electron diffraction (MicroED), the sample to be studied is a small chemical that is usually found in the form of a powder. The powder is applied directly to the EM grid to maintain a crystalline form. The data collection is then performed in diffraction mode, as shown by Martynowycz and Gonen¹⁰. In contrast, whole cells are too thick for the cryo-EM analysis. To address this, Bisson et al. report a method to use a SEM to physically drill into such a sample, in order to obtain thin sections suitable for structural studies¹¹.

From sample to structure, it is important to keep a record for each step of the process as many parameters are changed for each protein sample from protein concentration to microscope settings and data analysis. To this end, Wypych et al.¹² have developed a web-based information management tool that allows users to organize their workflow and keep track of all the details needed for future publications. This is particularly critical as automation is becoming more and more prevalent in cryo-EM, particularly in the industry settings.

Cryo-EM has advanced at an outstanding pace in the last decade, on both the hardware and software fronts. To ensure the widespread dissemination of best practices, and the wide adoption of new methods, a transparent set of protocols are necessary. This special issue will contribute to this effort.

JB acknowledges funding from BBSRC (BB/R009759/2) and HFSP (RGY0080/2021).