Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

The focus in protein folding has been very much on the protein backbone and sidechains. However, hydration waters make comparable contributions to the structure and energy of proteins. The coupling between fast hydration dynamics and protein dynamics is considered to play an important role in protein folding. Fundamental questions of protein hydration include, how far out into the solvent does the influence of the biomolecule reach, how is the water affected, and how are the properties of the hydration water influenced by the separation between protein molecules in solution? We show here that Terahertz spectroscopy directly probes such solvation dynamics around proteins, and determines the width of the dynamical hydration layer. We also investigate the dependence of solvation dynamics on protein concentration. We observe an unexpected nonmonotonic trend in the measured terahertz absorbance of the five helix bundle protein * 6 -85 as a function of the protein: water molar ratio. The trend can be explained by overlapping solvation layers around the proteins. Molecular dynamics simulations indicate water dynamics in the solvation layer around one protein to be distinct from bulk water out to Ϸ10 Å. At higher protein concentrations such that solvation layers overlap, the calculated absorption spectrum varies nonmonotonically, qualitatively consistent with the experimental observations. The experimental data suggest an influence on the correlated water network motion beyond 20 Å, greater than the pure structural correlation length usually observed.solvation dynamics ͉ THz spectroscopy ͉ lambda repressor ͉ molecular modeling W ater molecules interact with proteins on many length and time scales. Although the dynamics of the hydration water occurs on the picosecond time scale, ''slaving'' to fast solvent modes profoundly affects the slower but larger-scale protein motions (1). In return the protein influences the structure and dynamics of surrounding water molecules (2). X-ray crystallography has revealed ordered water structure around polar and charged sidechains (3), as well as cooperative insertion of water into hydrophobic cavities (4). Dielectric spectroscopy extends the time scale from microseconds down to 0.1 ns (5). Experiments have been extended to the THz range in films and crystals, probing motions on the picosecond time scale (6, 7). Hydrated protein powders probed by inelastic neutron scattering (0.1-100 ps) or solid-state NMR (nanoseconds) reveal that slower protein time scales and faster solvent time scales indeed show correlated dynamics (8). On the fastest time scales, 2D infrared spectroscopy and fluorescence of surface residues provide local probes of the dynamics in the femtosecond to picosecond range (9, 10). Coupling of modeling with experiments has revealed complex solvation structure around small biomolecules (11, 12), bridging our microscopic structural and thermodynamic understanding of biosolvation.Terahertz absorption spectroscopy of biomolecules fully solvated in water yields direct informa...

Passive macromolecular diffusion through nuclear pore complexes is thought to decrease dramatically beyond ∼40 kD. Using time-resolved fluorescence microscopy and Brownian dynamics simulations, Timney et al. show that this barrier is in fact much softer, decreasing along a continuum.