Explore the vast range of titles available.

Cellular membranes are formed from a chemically diverse set of lipids present in various amounts and proportions. A high lipid diversity is universal in eukaryotes and is seen from the scale of a membrane leaflet to that of a whole organism, highlighting its importance and suggesting that membrane lipids fulfil many functions. Indeed, alterations of membrane lipid homeostasis are linked to various diseases. While many of their functions remain unknown, interdisciplinary approaches have begun to reveal novel functions of lipids and their interactions. We are beginning to understand why even small changes in lipid structures and in composition can have profound effects on crucial biological functions.

Cryogenic electron microscopy (cryo-EM) is widely used to study biological macromolecules that comprise regions with disorder, flexibility or partial occupancy. For example, membrane proteins are often kept in solution with detergent micelles and lipid nanodiscs that are locally disordered. Such spatial variability negatively impacts computational three-dimensional (3D) reconstruction with existing iterative refinement algorithms that assume rigidity. We introduce non-uniform refinement, an algorithm based on cross-validation optimization, which automatically regularizes 3D density maps during refinement to account for spatial variability. Unlike common shift-invariant regularizers, non-uniform refinement systematically removes noise from disordered regions, while retaining signal useful for aligning particle images, yielding dramatically improved resolution and 3D map quality in many cases. We obtain high-resolution reconstructions for multiple membrane proteins as small as 100 kDa, demonstrating increased effectiveness of cryo-EM for this class of targets critical in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package. Membrane proteins exhibit spatial variation in rigidity and disorder, which poses a challenge for traditional cryo-EM reconstruction algorithms. Non-uniform refinement accounts for this spatial variability, yielding improved 3D reconstruction quality even for small membrane proteins.

Key Points Cellular membranes are laterally heterogeneous and consist of transient and dynamic domains with varying properties, which prominently include ordered lipid-driven domains that are referred to as lipid (or membrane) rafts. Membrane domains can be induced and regulated by a variety of interactions, which include specific lipid–lipid and lipid–protein interactions, bulk membrane properties, and interactions between membrane components and the underlying cytoskeleton. Advanced microscopy and biochemistry techniques facilitate the study of membrane domains; however, these domains still elude direct in vivo visualization. The multiplicity of possible organizational states and their context-dependent nature most likely account for experimental inconsistencies. Membrane rafts potentially have crucial physiological roles across cell types that range from immune cells to cancer cells. Membrane domains are conserved throughout the domains of life, which supports their functional importance in biological systems. Lipid rafts are relatively ordered membrane domains that are enriched in cholesterol and saturated lipids, and selectively recruit other lipids and proteins. They are dynamic and heterogeneous in composition and are thus challenging to visualize in vivo . New technologies are providing novel insights into the formation, organization and functions of these membrane domains. Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo , owing primarily to the development of improved biochemical and biophysical technologies.

Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches 1 – 6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations 7 – 14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems. The authors introduce a single-molecule DNA-barcoding method, resolution enhancement by sequential imaging, that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents.

The authors report a 2.6 Å resolution crystal structure of the human CB1 cannabinoid receptor trapped in the inactive conformation and bound to the antagonist taranabant. CB1 cannabinoid receptor structure The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the responses to endocannabinoids and the plant cannabinoid Δ 9 -tetrahydrocannabinol (THC). They are important drug discovery targets because of the therapeutic potential of receptor modulators for controlling disorders such as pain, epilepsy and obesity. Daniel Rosenbaum and colleagues determine a crystal structure of the human CB1 receptor bound to the inhibitor taranabant. The extracellular surface of the receptor is distinct from other lipid-activated GPCRs and forms a critical part of the ligand-binding pocket. Docking studies demonstrate how this pocket might accommodate tetrahydrocannabinol. The structure should aid drug discovery efforts for novel cannabinoid system modulators as potential therapeutics. The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) and to the widely consumed plant phytocannabinoid Δ 9 -tetrahydrocannabinol (THC) 1 . The cannabinoid receptors have been the targets of intensive drug discovery efforts, because modulation of these receptors has therapeutic potential to control pain 2 , epilepsy 3 , obesity 4 , and other disorders. Although much progress in understanding the biophysical properties of GPCRs has recently been made, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. Here we report the use of GPCR engineering and lipidic cubic phase crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6-Å resolution. We found that the extracellular surface of CB1, including the highly conserved membrane-proximal N-terminal region, is distinct from those of other lipid-activated GPCRs, forming a critical part of the ligand-binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function and will aid the design and optimization of therapeutic modulators of the endocannabinoid system.

Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-G i signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in G i coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs. Formylpeptide receptors (FPRs) are a class of chemotactic G protein-coupled receptors (GPCRs) that recognize pathogen- and host-derived formylpeptides. Here the authors report the 3.17 Å cryo-EM structure of the human FPR2-G i signaling complex with a bound peptide agonist and in combination with computational docking and MD simulations provide mechanistic insights into formylpeptide recognition by FPRs.

Macroautophagic clearance of cytosolic materials entails the initiation, growth and closure of autophagosomes. Cargo triggers the assembly of a web of cargo receptors and core machinery. Autophagy-related protein 9 (ATG9) vesicles seed the growing autophagosomal membrane, which is supplied by de novo phospholipid synthesis, phospholipid transport via ATG2 proteins and lipid flipping by ATG9. Autophagosomes close via ESCRT complexes. Here, we review recent discoveries that illuminate the molecular mechanisms of autophagosome formation and discuss emerging questions in this rapidly developing field. In this Review, Hurley and colleagues cover the most recent discoveries and the emerging molecular understanding of the mechanisms of autophagosome formation.

Eukaryotic plasma membranes are compartmentalized into functional lateral domains, including lipid-driven membrane rafts. Rafts are involved in most plasma membrane functions by selective recruitment and retention of specific proteins. However, the structural determinants of transmembrane protein partitioning to raft domains are not fully understood. Hypothesizing that protein transmembrane domains (TMDs) determine raft association, here we directly quantify raft affinity for dozens of TMDs. We identify three physical features that independently affect raft partitioning, namely TMD surface area, length, and palmitoylation. We rationalize these findings into a mechanistic, physical model that predicts raft affinity from the protein sequence. Application of these concepts to the human proteome reveals that plasma membrane proteins have higher raft affinity than those of intracellular membranes, consistent with raft-mediated plasma membrane sorting. Overall, our experimental observations and physical model establish general rules for raft partitioning of TMDs and support the central role of rafts in membrane traffic. Lipid rafts are plasma membrane domains that specifically recruit particular proteins. Here, the authors show that the surface area, length and palmitoylation of single-pass transmembrane domains are crucial for raft partitioning and propose a general model to predict protein association with rafts.

Polar auxin transport is unique to plants and coordinates their growth and development 1 , 2 . The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport 3 , 4 ; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N -1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold 5 . In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development. Structures of the Arabidopsis thaliana auxin exporter PIN1 in the apo state, bound to the natural auxin or bound to an inhibitor provide insights into the polar auxin transport mechanisms mediated by PIN family transporters.

Camelid single-domain antibody fragments (‘nanobodies’) provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.