The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre 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, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

The transporter associated with antigen processing (TAP) is an ATP-binding cassette (ABC) transporter essential to cellular immunity against viral infection. Some persistent viruses have evolved strategies to inhibit TAP so that they may go undetected by the immune system. The herpes simplex virus for example evades immune surveillance by blocking peptide transport with a small viral protein ICP47. In this study, we determined the structure of human TAP bound to ICP47 by electron cryo-microscopy (cryo-EM) to 4.0 Å. The structure shows that ICP47 traps TAP in an inactive conformation distinct from the normal transport cycle. The specificity and potency of ICP47 inhibition result from contacts between the tip of the helical hairpin and the apex of the transmembrane cavity. This work provides a clear molecular description of immune evasion by a persistent virus. It also establishes the molecular structure of TAP to facilitate mechanistic studies of the antigen presentation process.

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates the function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0 Å resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with an RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5' nucleotide.

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

The human cofactor complexes ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation) mediate activator-dependent transcription in vitro. Although these complexes share several common subunits, their structural and functional relationships remain unknown. Here, we report that affinity-purified ARC consists of two distinct multisubunit complexes: a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate substantial differences in size and shape between ARC-L and CRSP. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.

Primary cilia are sensory organelles present in many cell types. Based on an array of microtubules termed axoneme they form a specialized membrane compartment partaking in various signaling processes. Primary cilia of pancreatic islet beta cells play a role in autocrine and paracrine signaling and are linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of complete mouse and human beta cell cilia, revealing a disorganized 9+0 axoneme structure. Within the islet cilia are spatially confined within deep ciliary pockets or squeezed into narrow extracellular spaces between adjacent cells. Beta and alpha cell cilia physically interact with neighboring islet cells pushing and strongly bending their plasma membranes. Furthermore, beta cells can contain multiple cilia that can meet with other islet cell cilia in the extracellular space. Additionally, beta cell cilia establish connections with islet-projecting nerves. These findings highlight the pivotal role of beta cell primary cilia in islet cell connectivity, pointing at their potential functional role in integrating islet intrinsic and extrinsic signals. These novel insights contribute to understanding their significance in health and diabetes.

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

Comparing local neural structures across large sets of examples is crucial when studying gene functions, and their effect in the Drosophila brain. The current practice of aligning brain volume data to a joint reference frame is based on the neuropil. However, even after alignment neurons exhibit residual location and shape variability that, together with image noise, hamper direct quantitative comparison and retrieval of similar structures on an intensity basis. In this paper, we propose and evaluate an image-based retrieval method for neurons, relying on local appearance, which can cope with spatial variability across the population. For an object of interest marked in a query case, the method ranks cases drawn from a large data set based on local neuron appearance in confocal microscopy data. The approach is based on capturing the orientation of neurons based on structure tensors and expanding this field via Gradient Vector Flow. During retrieval, the algorithm compares fields across cases, and calculates a corresponding ranking of most similar cases with regard to the local structure of interest. Experimental results demonstrate that the similarity measure and ranking mechanisms yield high precision and recall in realistic search scenarios.

Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.