This EMBO Practical Course aims to teach participants how to integrate structural biology approaches to expedite characterization of large macromolecular complexes from the atomic to cellular scale. Lectures and practical sessions will explain the techniques used to produce, purify, reconstitute and characterize multi-subunit protein and protein/nucleic acid complexes for structural analysis. Specifically, this EMBO Practical Course will cover multi-subunit protein expression in bacteria, insect and mammalian cells; production, folding, and purification of non-coding RNAs; affinity, electrophoretic and centrifugation methods to isolate and purify sub-complexes and holocomplexes; biochemical and biophysical experiments that inform on the stoichiometric and conformational homogeneity of complexes; and strategies for determining the overall 3D architecture of large macromolecular assemblies and ultimately their structure at atomic or pseudo-atomic resolution.