Highlights d A comprehensive model is presented of the yeast nuclear pore complex (NPC) d Connectors link together different structural and functional layers in the NPC d Multiple structural and functional NPC isoforms co-exist in each cell d Modular construction allows structural plasticity and inner ring dilation of the NPC

Background: Ciliary microtubules contain hyperstable Ribbons of adjoining protofilaments.Results: Using echinoderm flagella, the locations of Ribbons, tektins, and Ca2+-binding proteins (related to human epilepsy) are studied biochemically and by immuno-cryo-electron tomography.Conclusion: The locations of these proteins create a biochemically, structurally unique region of ciliary A-microtubules.Significance: The results indicate specialized functions for Ribbons, with potential roles in assembly, motility, and/or signal transduction.

Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

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mTORC1 controls cellular processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by Ragulator. The Rag GTPases receive amino acid signals from upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binding to the Rag GTPases occurs via either of two modes: an inhibitory mode that has low enzymatic activity but high affinity, and a GAP mode that has high enzymatic activity but low affinity. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo‐EM structural models of the GATOR1‐Rag‐Ragulator complex, with the Rag‐Ragulator subcomplex occupying the inhibitory site, the GAP site, and both sites simultaneously. These structural models, together with the spatial constraints from the lysosomal membrane, reveal how GATOR1 coordinates the nucleotide loading states of both Rag subunits to transmit amino acid signals.

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