Cryo-electron microscopy (cryo-EM), the structural analysis of samples embedded in vitreous ice, is a powerful approach for determining three-dimensional (3D) structures of biological specimens. Over the past two decades, this technique has been used to successfully calculate subnanometer (<10 Å) resolution and, in some cases, near-atomic resolution structures of highly symmetrical and stable complexes such as icosahedral viruses and ribosomes, as well as samples that form ordered two-dimensional or helical arrays. However, determining high-resolution 3D structures of smaller, less symmetrical, and dynamic samples remains a significant challenge. The recent development of electron microscopes with automated data collection capabilities and robust direct electron detection cameras, as well as new powerful image processing algorithms, has dramatically expanded the number of biological macromolecules amenable for study using cryo-EM. In addition, these new technological and computational developments have been used to successfully determine <5 Å resolution 3D structures of samples, such as membrane proteins and complexes with either low or no symmetry, that traditionally were not considered promising candidates for high-resolution cryo-EM. With these exciting new advances, cryo-EM is now on pace to determine atomic resolution 3D structures.