Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. We describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic N-and C-termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We could therefore build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions. KeywordsAquaporin-0; lens; MIP; two-dimensional crystal; lipid-protein interaction; electron crystallography Members of the aquaporin (AQP) family form membrane pores that are either highly selective for water (aquaporins) or also permeable to other small neutral solutes such as glycerol and urea (aquaglyceroporins) (reviewed in 1 ). Structural studies have revealed that all AQPs share the same basic architecture, which consists of two tandem repeats, each containing a bundle of three transmembrane α-helices and a hydrophobic loop with the highly conserved asparagine-proline-alanine (NPA) motif 2 -8 . The two NPA-containing loops B and E fold back into the membrane and form short α-helices (HB and HE) that line the water pore. The ar/R constriction site, so named because it is formed by an aromatic and an arginine residue, confers water selectivity to AQP pores, while the NPA motifs play an important role in the proton exclusion mechanism (reviewed in 9 ).Correspondence to: Thomas Walz.Correspondence and requests for materials should be addressed to T.W. (twalz@hms.harvard.edu). Coordinates and structure factors for junctional and non-junctional AQP0 have been deposited in the Protein Data Bank (accession codes 2B6O and 2B6P, respectively).. Suplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. AQP0 is the most abundant protein in lens fibre cell membranes, where it forms not only water pores but also the 11-13 nm "thin lens junctions" that assemble upon proteolytic cleavage of the cytoplasmic termini 10 , 11 . We recently presented the structure of the AQP0-mediated membrane junction at 3 Å resolution as determined by electron crystallography of doublelayered two-dimensional (2D) crystals 7 . The structure showed that AQP0 junctions are stabilised by specific interactions between tetramers in adjoining membranes involving almost exclusively proline residues. Calculated pore profiles also showed that the pore in junctional AQP0 is highly constricted due to a substantially ...

Subcellular membrane-less assemblies are a reinvigorated area study in biology with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces we determined atomic structures of five segments of protein low-complexity domains associated with membrane-less assemblies. Their common structural feature is the stacking of segments into kinked β-sheets which pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic sidechains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, known to form networks and localize to membrane-less assemblies.

Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling two-component 120-subunit icosahedral protein nanostructures with molecular weights (1.8–2.8 MDa) and dimensions (24–40 nm diameter) comparable to small viral capsids. Electron microscopy, SAXS, and X-ray crystallography show that ten designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of independently purified components reveals rapid assembly rates comparable to viral capsids and enables controlled packaging of molecular cargo via charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines inspire the development of methods to engineer novel self-assembling protein structures. Although there has been exciting recent progress in this area, designing multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we address this challenge by developing a general computational method for designing protein nanomaterials in which two distinct types of subunits coassemble to a target symmetric architecture. We use the method to design five novel 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures, and experimentally demonstrate that the structures of the materials are in close agreement with the computational design models. The accuracy of the method and the universe of two-component materials that it makes accessible pave the way for the construction of functional protein nanomaterials tailored to specific applications.

Summary The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson’s disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears responsible for amyloid formation and cytotoxicity of α-synuclein. Here we report crystals of NACore having dimensions smaller than the wavelength of visible light and thus invisible by optical microscopy. Thousands of times too small for structure determination by synchrotron x-ray diffraction, these crystals have yielded an atomic resolution structure by the frontier method of Micro-Electron Diffraction. The 1.4 Å resolution structure demonstrates for the first time that this method can determine previously unknown protein structures and here yields the highest resolution achieved by any cryo-electron microscopy method to date. The structure reveals protofibrils built of pairs of face-to-face β-sheets. X-ray fiber diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, opening opportunities for design of inhibitors of α-synuclein fibrils.

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. Here we use trimeric protein building blocks to design a 24-subunit, 13 nm diameter complex with octahedral symmetry and two related variants of a 12-subunit, 11 nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5, for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery6, vaccine design7, and synthetic biology8.