CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches

Abstract

Sample preparation can present a significant hurdle within single particle cryo-electron microscopy (cryoEM), resulting in issues with reproducibility, data quality or an inability to visualise the sample. There are several factors which can influence this, including sample or buffer composition, grid type, route of sample preparation and interactions with the air-water interface (AWI). Here, we review some of the current routes for sample preparation and the associated challenges. We discuss a range of approaches for overcoming these challenges, such as minimising the grid preparation time, surfactants, grid type and biochemical approaches such as nanomagnetic beads. Finally, we discuss how a set of commercially available protein samples may serve as a benchmark suite for future technologies. This provides a route to compare techniques' abilities not just to generate high-resolution structures but also to overcome the challenges traditionally associated with cryoEM. As the field continues to produce new approaches to sample preparation and we start to better understand the underlying principles behind the behaviour of proteins within a thin film and in response to different environments, especially grid composition, it is hoped that more universal solutions can be provided that make the intractable systems tractable, improve resolution and, importantly, speed up data collection and reduce the currently required dataset sizes.

Keywords: cryo-electron microscopy; sample preparation; single particle cryoEM.

Figure 1.. Summary of the approaches taken to mitigate cryoEM sample preparation issues.

The protein sample (HA trimer, PDB 7VDF) is shown within the liquid thin film. The film's boundaries, representing the AWI, are denoted by two horizontal blue lines. The panels (left to right) represent adsorption to the AWI during the blotting process, modifying the AWI to reduce interactions with the AWI, sequestering the sample away from the AWI through a support film, reducing the interactions through faster sample preparation on millisecond timescales and modifying the sample to change its properties and interactions with the AWI. This figure was created with BioRender.com.