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Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1–GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo–electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.

Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function. X-ray and cryo-electron microscopy structures of fungal mitochondrial calcium uniporter proteins reveal a tetrameric architecture and shed light on the function of the channel.

In neurons, the loading of neurotransmitters into synaptic vesicles uses energy from proton-pumping vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases). These membrane protein complexes possess numerous subunit isoforms, which complicates their analysis. We isolated homogeneous rat brain V-ATPase through its interaction with SidK, a Legionella pneumophila effector protein. Cryo–electron microscopy allowed the construction of an atomic model, defining the enzyme’s ATP:proton ratio as 3:10 and revealing a homolog of yeast subunit f in the membrane region, which we tentatively identify as RNAseK. The c ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR, the latter of which is the (pro)renin receptor that, in other contexts, is involved in both Wnt signaling and the renin-angiotensin system that regulates blood pressure. This structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme’s catalytic and membrane regions.

Most membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes docked at the translocon, a heterogeneous ensemble of transmembrane factors operating on the nascent chain 1 , 2 . How the translocon coordinates the actions of these factors to accommodate its different substrates is not well understood. Here we define the composition, function and assembly of a translocon specialized for multipass membrane protein biogenesis 3 . This ‘multipass translocon’ is distinguished by three components that selectively bind the ribosome–Sec61 complex during multipass protein synthesis: the GET- and EMC-like (GEL), protein associated with translocon (PAT) and back of Sec61 (BOS) complexes. Analysis of insertion intermediates reveals how features of the nascent chain trigger multipass translocon assembly. Reconstitution studies demonstrate a role for multipass translocon components in protein topogenesis, and cells lacking these components show reduced multipass protein stability. These results establish the mechanism by which nascent multipass proteins selectively recruit the multipass translocon to facilitate their biogenesis. More broadly, they define the ER translocon as a dynamic assembly whose subunit composition adjusts co-translationally to accommodate the biosynthetic needs of its diverse range of substrates. Biochemical reconstitution and functional analysis reveal how newly synthesized multipass membrane proteins dynamically remodel the translocon to facilitate their successful biogenesis.

Actin depolymerizing factor (ADF) and cofilin accelerate actin dynamics by severing and disassembling actin filaments. Here, we present the 3.8 Å resolution cryo-EM structure of cofilactin (cofilin-decorated actin filament). The actin subunit structure of cofilactin (C-form) is distinct from those of F-actin (F-form) and monomeric actin (G-form). During the transition between these three conformations, the inner domain of actin (subdomains 3 and 4) and the majority of subdomain 1 move as two separate rigid bodies. The cofilin–actin interface consists of three distinct parts. Based on the rigid body movements of actin and the three cofilin–actin interfaces, we propose models for the cooperative binding of cofilin to actin, preferential binding of cofilin to ADP-bound actin filaments and cofilin-mediated severing of actin filaments. Cofilin is a small actin-binding protein that accelerates actin turnover by disassembling actin filaments. Here the authors present the 3.8 Å cryo-EM structure of a cofilin-decorated actin filament and discuss mechanistic implications.

AlphaFold can predict the structures of monomeric and multimeric proteins with high accuracy but has a limit on the number of chains and residues it can fold. Here we show that a combination of AlphaFold and all-atom symmetric docking simulations enables highly accurate prediction of the structure of complex symmetrical assemblies. We present a method to predict the structure of complexes with cubic – tetrahedral, octahedral and icosahedral – symmetry from sequence. Focusing on proteins where AlphaFold can make confident predictions on the subunit structure, 27 cubic systems were assembled with a median TM-score of 0.99 and a DockQ score of 0.72. 21 had TM-scores of above 0.9 and were categorized as acceptable- to high-quality according to DockQ. The resulting models are energetically optimized and can be used for detailed studies of intermolecular interactions in higher-order symmetrical assemblies. The results demonstrate how explicit treatment of structural symmetry can significantly expand the size and complexity of AlphaFold predictions. Current methods to predict structures of proteins cannot handle large assemblies with complex symmetries. Here, the authors demonstrate that structures of proteins with cubic symmetries can be accurately predicted with a method combining AlphaFold with symmetrical assembly simulations.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of L-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.

Bridge-like lipid-transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane-contact sites and are thought to mediate the bulk transfer of lipids from a donor membrane, typically the endoplasmic reticulum, to an acceptor membrane, such as that of the cell or an organelle 1 . Although BLTPs are fundamentally important for a wide array of cellular functions, their architecture, composition and lipid-transfer mechanisms remain poorly characterized. Here we present the subunit composition and the cryogenic electron microscopy structure of the native LPD-3 BLTP complex isolated from transgenic Caenorhabditis elegans . LPD-3 folds into an elongated, rod-shaped tunnel of which the interior is filled with ordered lipid molecules that are coordinated by a track of ionizable residues that line one side of the tunnel. LPD-3 forms a complex with two previously uncharacterized proteins, one of which we have named Spigot and the other of which remains unnamed. Spigot interacts with the N-terminal end of LPD-3 where lipids are expected to enter the tunnel, and experiments in multiple model systems indicate that Spigot has a conserved role in BLTP function. Our LPD-3 complex structural data reveal protein–lipid interactions that suggest a model for how the native LPD-3 complex mediates bulk lipid transport and provides a foundation for mechanistic studies of BLTPs. The LPD-3 complex structure reveals protein–lipid interactions that suggest a model for how the native LPD-3 complex mediates bulk lipid transport and provides a foundation for mechanistic studies of bridge-like lipid-transport proteins.

Mitochondrial complex III (CIII 2 ) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III 2 +IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII 2 , CIV, and SC III 2 +IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII 2 and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII 2 revealed long-range, coordinated movements across the complex, as well as the motion of CIII 2 ’s iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses. Most living things including plants and animals use respiration to release energy from food. Respiration requires the activity of five large protein complexes typically called complex I, II, III, IV and V. Sometimes these complexes combine to form supercomplexes. The complexes are similar across plants, animals and other living things, but there are also many differences. Detailed structures of the respiratory complexes have been determined for many species of animals, fungi and bacteria, highlighting similarities and differences between organisms, and providing clues as to how respiration works. Yet, there is still a lot to learn about these complexes in plants. To bridge this gap, Maldonado et al. used a technique called cryo electron microscopy to study the structure of complexes III and IV and the supercomplex they form in the mung bean. This is the first study of the detailed structure of these two complexes in plants. The results showed many similarities to other species, as well as several features that are specific to plants. The way the two complexes interact to form a supercomplex is different than in other species, as are several other, smaller, structural features. Further examination of complex III revealed that it is flexible and that movements are coordinated across the length of the complex. Maldonado et al. speculate that this may allow it to coordinate its role in respiration with its other cellular roles. Understanding how plant respiratory complexes work could lead to improvements in crop yields or, since respiration is required for survival, result in the development of herbicides that block respiration in plants more effectively and specifically. Further researching the structure of the plant respiratory complexes and supercomplexes could also shed light on how plants adapt to different environments, including how they change to survive global warming.