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Mycobacteria produce small molecules (mycobactins) to acquire the essential nutrient iron. Here, Earp et al determine the cryo-EM structures of the mycobactin exporter MmpL4, which also effluxes the TB drug bedaquiline.

Transmembrane E3 ligases are crucial in cellular homeostasis and metabolic regulation. Here, the authors provide the structural details of the ER-resident E3 ligase MARCH6/Doa10, uncovering its unique circular membrane structure and its role in ubiquitylation processes, essential for protein quality control.

Unconventional protein secretion emerges as an important mechanism in aggregate-prone protein removal. Here, the authors demonstrate that Plekhg5 mediates the unconventional secretion of Sod1 by presynaptic secretory autophagy.

Mycobacteria import life-essential iron using capturing molecules called mycobactins. In this work, the authors uncover how an ATP-fuelled transporter imports mycobactin across the inner membrane in an unusual “credit card” mechanism, during which mycobactin slides down a crevice at the transporter surface.

Although it has shown high efficacy in many tumor types, interleukin-12 (IL-12) treatment provokes severe systemic toxicity. Here, the authors discover that use of an IL-12Fc fusion cytokine with abolished binding to the neonatal Fc receptor presents efficacy in local glioblastoma therapy while preventing toxicity.

TM287/288 is a heterodimeric ABC transporter found in Thermotoga maritima. The crystal structure of TM287/288 in the inward-facing AMP-PNP–bound state provides new insight into nucleotide binding by heterodimeric transporters and will serve as an important model for eukaryotic disease-associated homologs.

Triacylglycerides help to secure the impermeability of mycobacterial cell envelope to some drugs. Here, authors solve the structure of the triacylglyceride transporter Rv1410 and proposed a molecular mechanism for triacylglyceride extraction.

Deep mutational scanning revealed the drug efflux activity profile of more than 1,430 single variants, enabling the identification of critical residues that regulate the activity of the bacterial drug efflux pump EfrCD in response to different drugs.

The mycobacterial ABC transporter IrtAB functions as a siderophore importer despite exhibiting an exporter fold in its structure, and contains a siderophore interaction domain capable of siderophore reduction and iron release inside the cell.

Oblique line scan microscopy achieves nanoscale spatial and sub-millisecond temporal resolution across a large field of view, enabling improved and robust single-molecule biophysical measurements and single-molecule tracking in both cells and solution.

ABC exporters hydrolyze ATP to pump substrates across membranes, but critical steps of the transport mechanism remain poorly understood. Here, the authors solve the crystal structure of outward-facing TM287/288 with the help of a state-specific sybody and gain insights into the role of the extracellular gate.

Elevator transporters, like SLC23 proteins, transport solutes by a mechanism involving a reorientation of two rigid domains. Here, the authors show that linkers connecting these domains control the carrier’s conformational space and thus its function.

Two thoroughly characterized nanobodies are sufficient to detect over 90% of clinical Escherichia coli isolates by targeting the conserved and abundant outer membrane protein OmpA.

Susac syndrome is an inflammatory pathology of the brain endothelium. Here the authors show that the pathology is driven by CD8 T cells attacking the endothelium, and that blocking T cell-endothelial adhesion ameliorates the disease in a mouse model, and associates with improved clinical score in 4 patients.

A method for protein binder selection and identification, NestLink, uses barcoding peptides detectable by mass spectrometry to select and biophysically characterize thousands of binders without requiring the handling of individual clones.

Lateral opening of the LptDE transporter in the outer membrane of Gram-negative bacteria is necessary for insertion of lipopolysaccharides. Here, Botte et al. report a cryo-EM structure of a partially opened LptDE transporter, in complex with rigid chaperones derived from nanobodies.

Whole-genome sequencing and phenotype data sharing are introduced in a national health system to streamline diagnosis and to discover coding and non-coding variants that cause rare diseases.

Here, the authors report the engineering, structural and biological characterization of synthetic nanobodies (sybodies) that display potent therapeutic activity against SARS-CoV-2 infection in animal models via targeting the virus receptor-binding domain.

Here, the authors isolate several nanobodies from a synthetic library that bind the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) and neutralize S pseudotyped viruses. Cryo-EM structure of Spike with one nanobody and further biophysical analysis shows competition with ACE2 binding.

Serial synchrotron crystallography reveals the structure of the human glycine transporter GlyT1, showing how a state-specific inhibitor exerts its effects, and potentially informing the design of new GlyT1 inhibitors to treat a range of disorders of the central nervous system.

A generally applicable approach to analyze intact membrane protein complexes by mass spectrometry is reported. This method allows subunit stoichiometry, lipid binding and the effects of post-translational modifications on complex formation to be explored.

Characterizing the total impulse response (TIR) of photoacoustic microscopes has been challenging due to difficulties distributing appropriate point sources. Here, the authors present a method for 3D generation of spatially-distributed optoacoustic point sources and show that subsequent TIR correction results in improved image quality.

Legionella pneumophila replicates in a Legionella-containing vacuole (LCV). Here the authors present the structure of the Legionella effector RidL N-terminal domain and reveal how RidL contributes to the subversion of retrograde trafficking by binding to the retromer coat complex subunit Vps29, which leads to a displacement of the regulator TBC1D5.

Volume-regulated anion channels (VRACs) are heteromers of LRRC8 proteins, all containing the obligatory subunit LRRC8A. Here, the authors develop and characterize nanobodies that bind LRRC8A and allosterically modulate the function of homomeric LRRC8A and endogenous heteromeric channels, hinting at functional mechanisms present in VRACs.

MFSD2A mediates uptake of the essential fatty acid DHA across the blood–brain barrier. Separately, via interactions with syncytin-2, MFSD2A contributes to the formation of the mother–fetus placental boundary. Cryo-EM analysis of a human MFSD2A–syncytin-2 complex provides new insights into how MFSD2A performs these dual roles.

Investigation of variation in three cohorts has identified multiple genetic signals associated with the pregnancy-specific liver disorder, intrahepatic cholestasis of pregnancy, giving insight into the disease which can cause preterm birth and stillbirth.

The authors show that loss-of-interaction with the nuclear importer, TNPO3, causes cytoplasmic mislocalization of RBM20 variants linked to severe cases of dilated cardiomyopathy. Restoring their nuclear localization alleviates the disease phenotype.

Whole-genome sequencing analysis of individuals with primary immunodeficiency identifies new candidate disease-associated genes and shows how the interplay between genetic variants can explain the variable penetrance and complexity of the disease.

Seeger et al. demonstrate concurrent 5-modal microscopy (Co5M), combining optoacoustic, two-photon, second and third harmonic generation, and brightfield microscopy in one system. Longitudinal changes associated with the wound healing process in mouse ears were monitored label-free in vivo using complementary information obtained from the Co5M system to characterize wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands.

This protocol describes in vitro procedures for generation of synthetic single-domain antibodies called ‘sybodies’. Sybodies can be engineered to target specific protein conformations, labile membrane proteins or protein complexes.

Mid-infrared optoacoustic microscopy enables label-free, bond-selective imaging in living cells

Trinco et al. measure aspartate uptake rates in proteoliposomes containing purified prokaryotic Na⁺-coupled aspartate transporter GltTk. To overcome limitation of protein orientation, they use synthetic nanobody that blocks transporters from outside and reveal mechanistic features of Na⁺-aspartate symport that cannot be observed in detergent solution.

In this work, the authors demonstrate that a coral thermostable GFP (TGP) is an improved tag compared to conventional GFPs. In addition to reporting melting point stability at temperatures near 90 °C, its fusion also helps increase expression levels of test membrane proteins. They further generated synthetic nanobodies against TGP to facilitate purification.