The current status and ongoing development of 3D electron diffraction and microcrystal electron diffraction in macromolecular crystallography are reviewed.

The modification of a crystallographic molecular-replacement pipeline for use in fitting search models to cryo-EM maps is described.

A perspective is provided on the future development of real-space methods in cryo-EM.

Coma-corrected single-particle data collection on a JEOL CRYO ARM 300 is described. It is shown that data collection with a throughput exceeding 6000 images per day and an image shift of up to 7 µm produces a reconstruction of apoferritin to a resolution of 1.7 Å.

Sample preparation is a major limiting factor in cryo-electron microscopy (cryo-EM), particularly for small macromolecules under 100 kDa. Here, experiences in preparing kinesin-binding protein (KBP; 72 kDa) and KBP–kinesin motor domain (110 kDa) samples for cryo-EM are detailed, in particular some of the challenges and opportunities arising from surface interactions on the cryo-EM grid.

An overview of the Scipion plugin for the RELION software package is presented and various image-processing capabilities within the Scipion framework are discussed.

Sample thickness is a key parameter in single-particle cryo-electron microscopy. Determining the sample thickness before data acquisition allows the targeting of optimal areas and the maximization of the data-output quality of single-particle cryo-electron microscopy sessions. Scripts and optimized workflows for EPU and SerialEM are presented and are available as open source.