The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2: upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions, which result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. Despite the recent accumulation in pLGIC structures4–8, no clear unifying view has emerged on conformational transitions involved in channel gating. Here we report four cryo-EM structures of the full-length mouse 5-HT3 receptor, ranging from 3.2 Å to 4.5 Å resolution, obtained in complex with the anti-emetic drug tropisetron, with serotonin, with serotonin and a positive allosteric modulator. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures represent new conformations, including that of an open state and of two novel ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles; we characterize motions at the gate level and cation accessibility in the pore. Put together, the data deepen our understanding of the gating mechanism of pLGICs, while capturing ligand binding in unprecedented detail.

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.