This review describes how electron-microscopy methods have helped to reveal the structure of the ciliary axoneme.

Single-particle cryo-EM is a powerful technique to study the assembly process of the largest mitochondrial respiratory chain complex, NADH:ubiquinone oxidoreductase or complex I. Here, the new insights that have been gained into the molecular functions of assembly factors are reviewed.

To simplify the cryo-electron tomography workflow and make this technique more accessible to all researchers, the Tomo Live software has been developed, which performs on-the-fly reconstruction of tilt series, enabling real-time data-quality monitoring, curation and export.

Likelihood-based cryo-EM docking using the emplace_local software is faster and more sensitive than the related em_placement software when the approximate location of a component is known. It is conveniently available through a plugin to the ChimeraX visualization software.

The automation of cryoEM pipelines to aid data-quality analysis during acquisition poses a number of challenges, particularly at facilities where the expected data rates are high. The use of a modern service-based architecture deployed on Diamond Light Source's on-premises cloud computing cluster to tackle some of these challenges is demonstrated, focusing on the provision of an early-stage processing pipeline for electron tomography that produces first-attempt reconstructed volumes within minutes of tilt-series acquisition.

This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow, as well as a wide variety of case studies. This illustrates the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

relion_live.py and relion_analyse.py, two web-based tools to enhance cryo-EM data processing in RELION, are introduced, providing an interface for real-time feedback on data collection and simplified interpretation of metadata. Additionally, an analytical script for ice-quality estimation is provided, empowering researchers to make informed decisions to improve data quality and accessibility in cryo-EM.