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.

The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm its functional importance we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, suggesting this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a profound preference for tubulin from the same species.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.energy metabolism | phototransduction A significant energy distribution problem can arise from the relative locations of mitochondria, ion pumps, and synapses in neurons. In photoreceptors, ion pumps occupy the intervening space between the centrally located mitochondria and the synaptic terminal. Ion pumping in dark-adapted photoreceptors consumes ∼20× more energy than neurotransmission (1). Therefore, the pumps could intercept all the metabolic energy made by the mitochondria before it can reach the synaptic terminal. In the vascularized retinas of mice, rats, and humans (2-4) this problem is solved by the presence of additional mitochondria in the terminal. However, in the avascular retinas of zebrafish, salamanders, rabbits, and guinea pigs there are no mitochondria in the terminals (2, 4, 5), which creates a need to partition some of the energy made by the central mitochondria into a protected form that can bypass the ion pumps to support the essential energy demands of the synaptic terminal.Energy consumption within retinal photoreceptors is compartmentalized and light-dependent. During illumination, phototransduction and light adaptation consume energy in the outer segment (OS). In darkness, energy is consumed by ion pumps in the inner segment and by glutamate release at the synaptic terminal (1). Energy demands and O 2 consumption are far greater in darkness than in light (1, 6-8).Metabolic energy is distributed in most cells as either ATP or phosphocreatine (PCr). There are 2 isoforms of creatine kinase (CK) in neurons, ubiquitous mitochondrial creatine kinase (uMtCK), and brain-type cytoplasmic creatine kinase (CK-B). uMtCK creates PCr from ATP at mitochondria (9), and CK-B can recreate ATP from PCr at sites of energy demand. In this way uMtCK and CK-B can collaborate to transfer metabolic energy between neuronal compartments (10, 11). This paper descr...