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Version 1.2 of the software system, termed Crystallography and NMR system (CNS), for crystallographic and NMR structure determination has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure determination, (ii) to provide tools for structure solution of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromolecules and (iv) to integrate all sources of information into all stages of the structure determination process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallographic refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, especially at lower resolution, generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron density maps. Consistent with the modular design of CNS, these additions and changes were implemented in the high-level computing language of CNS.

The essential autophagy mediator ATG14 promotes vesicle fusion by forming homo-oligomers, which bind to a component of the SNARE membrane fusion complex and stabilize this complex on autophagosomes. Autophagy-specific membrane fusion During the process of autophagy, cells break down their own components and sequester them in specialized vesicles called autophagosomes, which eventually fuse with lysosomes for degradation of their content. Membrane fusion is an important event not only during early biogenesis of autophagosomal vesicles but also when autophagosomes unite with lysosomes. Qing Zhong and colleagues show here that the essential autophagy mediator ATG14 promotes vesicle fusion by forming homo-oligomers, which bind to a component of the SNARE membrane fusion complex and stabilize this complex on autophagosomes. Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation 1 , 2 . Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes 3 , 4 , 5 , 6 , 7 . However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex 8 , 9 , 10 , 11 , promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N -ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17–SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro . Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17–SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome–endolysosome fusion.

Synaptotagmin, complexin, and neuronal SNARE (soluble N -ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale. An atomic model of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex in neuronal exocytosis accounting for vesicle priming and cooperation in synchronizing and activating evoked release on the sub-millisecond timescale. Three-way complex primes synapses for membrane fusion For rapid neurotransmitter release upon the arrival of an action potential, synaptic vesicles are 'primed' to undergo synchronous fusion with the pre-synaptic membrane, but the molecular basis of such priming is unknown. Now, with two large co-crystal structures, Axel Brunger and colleagues reveal a new and unexpected three-way interface between the proteins synaptotagmin (Syt1), complexin (Cpx) and the SNARE complex, beside a previously identified primary interface involving another molecule of Syt1 with the same SNARE complex. Through a combination of mutagenesis, biochemistry and electrophysiology, the authors show how this tripartite interface locks the primed complex into a state of low fusion probability and how action-potential-driven Ca 2+ binds to the Syt1 molecules to unlock the complex, allowing full zippering of the SNARE complex and triggering membrane fusion in a highly synchronized fashion on the sub-millisecond timescale.

Membrane fusion triggered by Ca 2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes 1 – 4 . For neurotransmitter release, the Ca 2+ sensitivity is introduced by interactions between the Ca 2+ sensor synaptotagmin and the SNARE complex 5 , and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion 6 . Disruption of Ca 2+ -triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis 7 – 11 . Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca 2+ -triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca 2+ -binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca 2+ -triggered fusion at physiological Ca 2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca 2+ -triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways. Peptides that disrupt Ca 2+ -triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca 2+ - and Mg 2+ -bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca 2+ -independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca 2+ -triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca 2+ -triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca 2+ triggering, moves en bloc as Ca 2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces. The first crystal structures of complexes between synaptotagmin-1 and neuronal SNARE, bound to either Ca 2+ or Mg 2+ , are described, and show that Ca 2+ -triggered neurotransmitter release relies on a large, Ca 2+ -independent interface. Synaptotagmin–SNARE complex structures The transmembrane protein synaptotagmin-1 and neuronal SNARE proteins are key players in neurotransmitter release, but how they cooperate to trigger synaptic vesicle fusion in response to calcium signals remains unclear. Now Axel Brunger and collaborators report the first crystal structures of complexes between these proteins, bound to either Ca 2+ or Mg 2+ , and show that Ca 2+ -triggered neurotransmitter release relies on a large, Ca 2+ -independent interface. The work also introduces new techniques to collect diffraction data from an X-ray free-electron laser, allowing near-atomic resolution of a protein complex from small numbers of crystals.

Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin—SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion. NSF hydrolyzes ATP to disassemble SNARE complexes. Here, the authors find NSF colocalizes with syntaxin nanodomains, reveal disassembly of syntaxin oligomers and other pre-fusion cis-SNARE complexes by NSF, and show how sequential hydrolysis drives disassembly.

α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself. The central nervous system coordinates many different activities by sending instructions to large numbers of cells and, simultaneously, processing all the signals that are sent back to the brain. All these messages are carried by electrical pulses that travel along chains of neurons, with neurotransmitter molecules enclosed inside synaptic vesicles conveying the messages across the synapses between neurons. A protein called α-synuclein is thought to have a role in the transport of neurotransmitter molecules across synapses, but the details of its involvement are not fully understood. Mutations in the gene that codes for α-synuclein, and also duplications and triplications of this gene, are known to lead to an increased risk of early onset Parkinson's disease, a condition where the central nervous system degenerates. Moreover, the Lewy bodies found in the neurons of patients with Parkinson's disease contain high concentrations of α-synuclein. Again, however, none of this is fully understood. Diao et al. have shed new light on these questions by creating synthetic vesicles to mimic what happens in real synapses, and using optical microscopy to observe the behaviour of these vesicles. They found that native α-synuclein (and another set of membrane proteins) increases the availability of synthetic vesicles at the synapse by causing them to cluster together. In a second experiment, Diao et al. showed that native α-synuclein does not decrease calcium-triggered fusion between membranes, the process that releases neurotransmitter into the synaptic cleft. In contrast, it is known that pathogenic α-synuclein aggregates directly interfere with the release of the neurotransmitter molecules. Moreover, when Diao et al. used a particular mutant form of α-synuclein that is associated with Parkinson's disease, the vesicles did not form clusters. If these results are confirmed in vivo, the role played by native α-synuclein in the central nervous system, and the connection between α-synuclein and Parkinson's disease, will be much clearer.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell–cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca 2+ triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca 2+ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca 2+ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca 2+ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca 2+ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.