(International Tables for Crystallography Volume F, 2010, in press) Four-Dimensional Cryo Electron Microscopy at Quasi Atomic Resolution: "IMAGIC 4D"

The traditional tools of the structural biologist seeking to understand macro-molecules and their complexes are X-ray crystallography and NMR spectroscopy. Singleparticle cryo electron microscopy (“cryo-EM”) has established itself as a new structural biology technique over the last 15 years. Spectacular insights into the functioning of macromolecular complexes have been achieved especially from combining cryo-EM with the earlier approaches. The resolution levels achieved improved over the last decade from ~10Å to sometimes better than ~4Å, meaning that a de-novo structure determination based on single-particle cryo-EM studies alone is now feasible. More challenging is the new perspective that cryo-EM brings: sorting heterogeneous populations of molecules into individual 3D conformers resulting in sequences of related 3D structures, or short: "4D cryo-EM". Thanks to these developments, single-particle cryo-EM has become the technique of choice to shed light on the functioning of many a complex biological system. The design of the software instrumentation for 4D cryo-EM is crucial. In this paper we elaborate on organisation issues for single-particle cryo-EM software, as exemplified by recent developments in the “IMAGIC 4D” software system.