Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules. We provide an overview of the cryo-EM method and introduce contemporary studies to investigate biomolecular structure and dynamics, including examples from the recent literature. Cryo-EM is a powerful tool for the investigation of biological macromolecular structures including analysis of their dynamics by using advanced image-processing algorithms. The method has become even more widely applicable with present-day single particle analysis and electron tomography. The cryo-EM method can be used to determine the three-dimensional structure of biomacromolecules in near native condition at close to atomic resolution, and has the potential to reveal conformations of dynamic molecular complexes. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.