Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.

Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine–glycine–aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.

Characterising the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. In dsDNA and dsRNA viruses, the packaged double-stranded nucleic acid, constrained by the rigidity of the doublehelix, adopts a liquid crystalline arrangement 1-5. Owing to the different cellular fates of DNA and RNA, the life cycles of these viruses are strikingly dissimilar. Current models suggest that dsDNA viruses predominantly display single-spooled conformations due to the lack of genome segmentation and the absence of intracapsid transcriptional machinery 6-8. As dsRNA triggers host defence mechanisms if released into the cytoplasm 9 , dsRNA viruses retain their genomes within a core particle containing the enzymes required for RNA replication and transcription 10,11,12. Their genomes vary greatly in the degree of segmentation. In reoviruses (Reoviridae, 10-12 segments) the genome organizes in nonspooled fashion and is tightly associated with the viral RNA-dependent RNA polymerases (RdRPs) 11-14. However, whether this organization is generally applicable in dsRNA viruses remains unknown. Here, we use cryogenic electron microscopy (cryo-EM) to show that dsRNA viruses