Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

SUMMARY The physiology of N-Methyl-D-aspartate (NMDA) receptors in mammals is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino terminal domain (ATD). Recent crystal structures of GluN1/GluN2B NMDA receptors in the presence of agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here, we combine x-ray crystallography, single-particle electron cryomicroscopy, and electrophysiology to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open-conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

Integration of EM, protein–protein interaction, and phenotypic data reveals novel insights into the structure and function of the nuclear pore complex’s ∼600-kD heptameric Nup84 complex.

Background:Fascin is the main actin-bundling protein in filopodia. Results: Biochemical, cryo-electron tomographic, and x-ray crystal structural data reveal the unique actin-binding characteristics of fascin. Conclusion: There are two major actin-binding sites on fascin and there is a concerted conformational change between the actin-binding sites. Significance: These data will advance our understanding of the function of fascin in filopodial formation.

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(1,2) indicates that the differences between strains must somehow be embodied in distinguishable, self-propagating structural features of the infectious amyloid fibrils formed by seeded growth in vitro with recombinant Sup35 prion protein. Induction of prion disease in transgenic mice by injection of amyloid aggregates of recombinant mouse prion protein (3) supports the hypothesis (4) that strains of the mammalian transmissible spongiform encephalopathies are caused by self-propagating misfolded forms of the prion protein.Considering all of the evidence associating both mammalian and yeast prions with infectious amyloid fibers, and the many atomic models proposed for amyloid structures (cf. refs. 5 and 6) since the cross-␤ polypeptide folding pattern (7, 8) was recognized as characteristic of pathological amyloid fibers (9, 10), it is surprising that molecular explanations of how amyloid fibers are actually constructed and why they are so stable remain elusive.Seeded amyloid fibril growth from soluble protein interacting with nucleating amyloid fragments (which is the presumed mechanism of prion propagation in vivo) was first demonstrated in vitro with fibrous insulin (11) before its cross-␤ conformation was identified (12). Now such self-nucleated growth is considered a defining characteristic of amyloid fiber assembly (cf. refs. , which we have determined, are comparable. Correlation of thermal instability with dominance and suppressor strength implies that greater structural lability of infectious amyloid fibrils allows their more efficient fragmentation by cellular machinery to generate the nuclei that specifically sop up Sup35 molecules, thereby selectively propagating the more frangible fibrils.The molecular architecture of the amyloid fibrils formed in our studies (1) by the recombinant Sup35 prion domain fused with GFP is similar to that of in vitro self-assembled Ure2p constructs that have been analyzed in a detailed electron microscopy study by Baxa et al. (23). They established that the N-terminal 70-residue prion-defining domain of this yeast prion protein (17) forms the amyloid core and the globular C-terminal portion could be replaced in fusion constructs by four other globular proteins, including GFP, with no evident effect on the amyloid core structure. Mass per length (mpl) measurements by scanning transmission electron microscopy (STEM) (23) indicated that, within the experimental uncertainty, there might be just one prion molecule for each 4.7-Å cross-␤ repeat period of the amyloid core, independently of the size of the attached C-terminal domain. These results suggested a simple model for the amyloid core in which segments of a sinuously folded N-terminal domain should form a single layer of regularly Abbreviations: STEM, scanning transmission electron microscopy; TEM transmission electron microscope; mpl, mass per length; TMV, tobacco mosaic virus; X-␤ ply, cross-␤ ply.

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To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.