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.

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.

Although several different flaviviruses may cause encephalitis, Japanese encephalitis virus is the most significant, being responsible for thousands of deaths each year in Asia. The structural and molecular basis of this encephalitis is not fully understood. Here, we report the cryo-electron microscopy structure of mature Japanese encephalitis virus at near-atomic resolution, which reveals an unusual “hole” on the surface, surrounded by five encephalitic-specific motifs implicated in receptor binding. Glu138 of E, which is highly conserved in encephalitic flaviviruses, maps onto one of these motifs and is essential for binding to neuroblastoma cells, with the E138K mutation abrogating the neurovirulence and neuroinvasiveness of Japanese encephalitis virus in mice. We also identify structural elements modulating viral stability, notably Gln264 of E, which, when replaced by His264 strengthens a hydrogen-bonding network, leading to a more stable virus. These studies unveil determinants of neurovirulence and stability in Japanese encephalitis virus, opening up new avenues for therapeutic interventions against neurotropic flaviviruses.

Significance: The negative-strand RNA viruses comprise several significant human, animal, and plant pathogens that have considerable health and economic impact globally. During infection, replication of the single-stranded negative-sense RNA genome occurs through a complementary RNA intermediate, which is believed to complex with viral proteins to form a complementary ribonucleoprotein (cRNP). The isolation of these complexes from infected cells has never been accomplished, greatly hampering our understanding of genome replication. We report a technological advance for the isolation of this elusive but essential component of the influenza A virus replication machine. Structural and functional characterization of the influenza A virus cRNP has led to the proposal of a model of genome replication that relies on a trans -activating viral RNA-dependent RNA polymerase.