Themed collection Challenges in biological cryo electron microscopy

CryoEM has made amazing progress during the past 10 years and is in a process of rapid transformation. This year’s Faraday Discussions examined some of the remaining challenges.

A brief overview of the highlights in the development of cryo EM is followed by an introduction to the discussion talks. The image represents a field of single particles with conformational and compositional heterogeneity, and preferred orientation.

Cryo-EM of samples prepared from mass-selected and soft-landed protein complexes creates a direct link between structure and complementary chemical information.

Experimental workflows combining mouse genetics, cryogenic correlated light and electron microscopy and cryo-electron tomography to bridge length scales from the whole organism to molecules.

In this study, we reflect on widely-used helical processing routines in cryoEM and suggest a new workflow capable of uncovering heterogeneities and complex symmetries that could otherwise be overlooked.

A microfluidic sample preparation method for TEM, which entirely avoids paper blotting and allows the passivation of the air–water interface during the preparation process, is presented.

First results from a new cryo-EM grid preparation method are presented, significantly faster than previous methods. The short time delay has the potential to improve grid preparation for challenging samples and to allow faster time-resolved EM.

Atomic models from cryo-EM show a preference for geometric quality with the expense of agreement with the map data. This study highlights the lack of validation standards and CCP-EM efforts and related developments in this context.

Modifications to a commercial plunge-freezer are described, enabling temperature-sensitive, light-sensitive and oxygen-sensitive samples to be frozen for analysis by cryo-TEM.

Current and future technology for in situ electron cryomicroscopy is considered from theoretical and practical viewpoints.

Cryo-electron microscopy (cryoEM) has been transformed over the last decade, with continual new hardware and software tools coming online, pushing the boundaries of what is possible and the nature and complexity of projects that can be undertaken.

Cryo-STEM tomography (CSTET) reveals the boundary of hemozoin crystals in situ within the digestive vacuole of P. falciparum.

AlphaFold2 predicts protein folds from sequence, which can be used for experimental structural biology, in construction and de novo protein design, prediction of complexes and perhaps even effects of mutations and conformational space exploration.

We explore the use of generalised scattering properties of biomolecules for restoration of contrast and visual improvement of cryo-EM maps.

A comparison of STA and SPA processed data acquired with/without super-resolution imaging mode enabled.

We, for the first time, reconstruct from the published incomplete cryo-EM density a complete MS2 virus at atomistic resolution, that is, the capsid with the genome, and validate the result by all-atom molecular dynamics with explicit water.

In cryo-tomography and subtomogram averaging, particle picking constitutes a major bottleneck and requires significant user time and expertise. Here we summarise particle-picking strategies, focusing in particular on membrane-associated particles.

A progressive CryoEM map validation scheme is presented. The scheme may include the map, half maps, 2D classes, particles, angles, coordinates, defoci, micrographs, and image processing workflow.

This paper presents new cryo-EM image processing methods for amyloids, including automated picking and strategies for helical structure determination in RELION. The image shows examples of tau filament structures that were solved using these methods.