Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a threedimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.cryoelectron microscopy | fatty acid synthesis | acyl carrier protein | molecular architecture

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca 2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca 2+ -triggered release. T he process of vesicle trafficking is central for transporting materials both inside and outside of cells and is essential for maintaining cellular homeostasis (1, 2). Synaptic vesicle fusion releases neurotransmitter molecules into the synaptic cleft upon an action potential-a fast (<5 ms) and highly regulated process. The neuronal SNARE proteins synaptobrevin-2/VAMP2 and syntaxin-1A are anchored in the synaptic vesicle and plasma membranes, respectively, whereas SNAP-25 is peripherally associated with the plasma membrane. The N-to C-terminal zippering of the trans SNARE complex between synaptic and plasma membranes provides the force for membrane juxtaposition and fusion (3), but SNAREs alone are not Ca 2+ -sensitive. Synaptotagmins are membrane-tethered proteins containing two Ca 2+ -binding C2 domains, and a subset of the 16 isoforms of mammalian synaptotagmins act as Ca 2+ sensors for neurotransmitter release: e.g., synaptotagmin-1 (Syt1) is the Ca 2+ sensor for evoked synchronous neurotransmitter release (4). The system is exquisitely fine-tuned to increase the probability of fusion between synaptic vesicles and the plasma membrane by orders of magnitude upon Ca 2+ binding to Syt1. Synaptotagmins simultaneously interact with anionic phospholipid membranes (5) and the neuronal SNARE complex (6, 7). The SNARE complex also interacts with complexin (Cpx), a small soluble protein that both activates evoked release and regulates spontaneous release (8, 9). Moreover, Cpx forms a tripartite complex with the SNARE complex and Syt1 (10). This tripartite complex is part of the prefusion, "primed" state of the synaptic vesicle fusion machinery. Both Munc13 and Munc18 are important for priming, and, at the molecular level, they facilitate proper SNARE complex assembly (11, 12).Although high-resol...

Fatty acid mega-synthases (FAS) are large complexes that integrate into a common protein scaffold all the enzymes required for the elongation of aliphatic chains. In fungi, FAS features two independent dome-shaped structures, each 3-fold symmetric, that serve as reaction chambers. Inside each chamber, three acyl-carrier proteins (ACP) are found double-tethered to the FAS scaffold by unstructured linkers; these are believed to shuttle the substrate among catalytic sites by a mechanism that is yet unknown. We present a computer-simulation study of the mechanism of ACP substrate-shuttling within the FAS reaction chamber, and a systematic assessment of the influence of several structural and energetic factors thereon. Contrary to earlier proposals, the ACP dynamics appear not to be hindered by the length or elasticity of the native linkers, nor to be confined in well-defined trajectories. Instead, each ACP domain may reach all catalytic sites within the reaction chamber, in a manner that is essentially stochastic. Nevertheless, the mechanism of ACP shuttling is clearly modulated by volume-exclusion effects due to molecular crowding and by electrostatic steering toward the chamber walls. Indeed, the probability of ACP encounters with equivalent catalytic sites was found to be asymmetric. We show how this intriguing asymmetry is an entropic phenomenon that arises from the steric hindrance posed by the ACP linkers when extended across the chamber. Altogether, these features provide a physically realistic rationale for the emergence of substrate-shuttling compartmentalization and for the apparent functional advantage of the spatial distribution of the catalytic centers.