Exploration of native cellular processes within biological specimens, structures of recombinant proteins with ligands and the molecular workings of complex macromolecular assemblies and machinery using cryo-electron microscopy (cryo-EM) is a multi-faceted and dynamic area of research. Advances in technology and improvements in methods for sample preparation, data collection, and processing are evolving processes where innovations continue to push the limits of resolution and discovery. Complementation with integrated and computational methods in structural biology further augments perspectives in answering biological questions. However, the accessibility of cryo-EM, both single-particle analysis and cryo-electron tomography, and its complementary methods for dissemination of practical details of methodology remain limited. This issue of Methods Collections aims to help address the challenge and add to the knowledge-base by highlighting protocols pertaining to:

1. Specific sample types that may have distinct requirements in preparation and structure determination

2. Established and innovative methods in all aspects of the cryo-EM structure solution pipeline including overcoming challenges, which inform discovery, especially in research areas of high interest 

3. Complementary methods that support or enhance cryo-EM investigations in structural biology.