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The asymmetric distribution of lipopolysaccharide (LPS) on the surface of the bacterial outer membrane is essential and crucial for antibiotic resistance. Here, authors characterize the LPS translocon holo-complex, LptDEMY, uncovering conformational translocon state transitions that might explain how LPS is assembled at the outer membrane surface.

Researchers have resolved cryo-EM structures of the bacterial TamAB machinery in two distinct states: a non-hybrid and a novel hybrid barrel. These structures suggest each state enables a distinct function, facilitating outer membrane protein folding and phospholipid transport, respectively.

Gram-negative bacteria use a multiprotein complex, LptB₂FGC, to transport lipopolysaccharides (LPS) to the outer membrane. Here, Fiorentino et al. present cryo-EM structures of the complex from Pseudomonas aeruginosa, revealing species-specific features and providing insights into LPS transport mechanisms.

Bacterial resistance to polymyxin antibiotics is conferred by enzymes such as phosphoethanolamine transferases, which add positively charged phosphoethanolamine to lipid A. Here, the authors present the structure of one such enzyme in its liganded form, and propose an enzymatic mechanism that may be generally applicable to other phosphoform transferases.

The study reveals the structure of ArnC, a membrane glycosyltransferase that modifies undecaprenyl phosphate with aminoarabinose (L-Ara4N), leading to polymyxin resistance. The structure of ArnC in two states and MD simulations provide insights regarding the catalytic cycle of the enzyme.

Here, the authors present cryoEM structures of AftB, a key mycobacterial enzyme that adds terminal arabinose residues to the cell wall. In concert with functional assays and MD simulations, mechanistic insights are presented.

Wzc is the master regulator of the biosynthesis and export of bacterial capsular polysaccharides and exopolysaccharides. Here, the authors report cryoEM structures of intermediate states and provide insights into phosphorylation and transmembrane signalling.

Here the authors investigate of the structural basis of substrate recognition and the catalytic mechanism of the mannosyltransferase PimE, which is crucial for the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs) in Mycobacterium tuberculosis.

This study reveals that an outer membrane protein from the predator Bdellovibrio bacteriovorus forms a pentameric assembly that traps a lipid monolayer within. This allows the discovery of two superfamilies, distributed across a wide range of bacteria, likely to adopt a similar architecture.

Native mass spectrometry, HDX-MS and MD simulations define the mechanism for how LPS binding to the Gram-negative outer membrane complex LptDE opens the LptD lateral exit gate and how thanatin impairs transport across the periplasm.

Gangliosides are bioactive lipids important for neuronal cell function. Here, authors reveal the dynamic structure of the ganglioside synthetic enzyme B4GALNT1 and demonstrate how it inserts into membranes to access and process lipid substrates.

Bacterial cell shape is dependent on the formation of the extracellular sugar polymer called peptidoglycan. Here the authors describe RodA-PBP2, the enzymatic core of the elongasome, which is the complex responsible peptidoglycan synthesis, and utilize an integrated approach to investigate the mechanism of peptidoglycan biosynthesis.

Bacterial O antigen polysaccharides play a role in innate immune response evasion. Here, the authors uncover the ATP-bound structure of the Aquifex aeolicus WzmWzt O antigen ABC transporter, shedding light onto the mechanism of lipid-linked polysaccharide translocation.

TMEM16K is a member of the TMEM16 family of integral membrane proteins that are either lipid scramblases or chloride channels. Here the authors combine cell biology, electrophysiology measurements, X-ray crystallography, cryo-EM and MD simulations to structurally characterize TMEM16K and show that it is an ER-resident lipid scramblase.

Cryo-electron microscopy structures of the bacterial O-antigen ligase WaaL, combined with genetics, biochemistry and molecular dynamics simulations, provide insight into the mechanism by which WaaL catalyses the biosynthesis of lipopolysaccharide.

In eukaryotes the SLC35 family of solute carriers mediate the selective uptake of nucleotide sugars from the cytoplasm into the Endoplasmic Reticulum or Golgi. Here authors report the crystal structure of the yeast GDP-mannose transporter, Vrg4, bound to guanine monophosphate (GMP) revealing the molecular basis for GMP recognition and transport.

Two crystal structures of the Escherichia coli β-barrel assembly machinery (BAM complex) are presented, one of which includes all five subunits (BamA–BamE), in two distinct conformational states; together with functional assays and molecular dynamics stimulations, these structures help to generate a model for outer membrane protein insertion.

Peptidoglycan biosynthesis is regulated by a feedback mechanism preventing the pathway from sequestering essential lipid carriers used to assemble and transport surface glycan subunits.

In this work, the authors present structural and functional evidence of lipid exchange within the gram-negative Mla pathway, broadening our understanding of lipid transport in bacteria and providing insight into the mechanism of a unique ABC transporter.

KdpFABC is a high-affinity bacterial K⁺ pump which combines the ion channel-like KdpA and the P-type ATPase KdpB. Here, the authors elucidate the mechanisms underlying transport and the coupling to ATP hydrolysis, and provide evidence that ions are transported via an intersubunit tunnel through KdpA and KdpB.

The Nitrosopumilus maritimus surface layer (S-layer) concentrates ammonium ions on its cell-facing side, acting as a multichannel sieve on the cell membrane.

Interpretation of lipid-like densities in cryo-EM structures of membrane proteins is challenging. Here authors present LipIDens, enabling molecular dynamics analysis of protein-lipid interactions.

Here, the authors identify a third component of the outer membrane LPS translocon in Escherichia coli called LptM. Biochemical analysis and structural modelling reveal that LptM binds the LPS translocon by mimicking its native substrate, so stabilising an active conformation of the complex.

Second messengers regulate a broad spectrum of processes in bacteria. Here, authors characterize how the second messenger cyclic di-AMP inhibits the potassium transporter KimA using cryo-EM, molecular dynamics simulations and functional assays.

Phospholipid scramblase 1 (PLSCR1), a protein induced by IFNγ, acts as a defence factor against SARS-CoV-2 and other coronaviruses by inhibiting the fusion of the virus with host-cell membranes.

Bacterial cell wall enzymes and their precursors are critical targets for antibiotic development. Here, the authors investigate several biosynthetic enzymes with their substrates and show that the passage of substrates and products in the pathway is controlled by their relative binding affinities.

CD59 protects human cells from damage by the MAC immune pore. The authors show how CD59 inhibits MAC, by deflecting pore-forming β-hairpins of complement proteins. As well as how the membrane environment influences the role of CD59 in complement regulation and in host-pathogen interactions.

A cryo-electron microscopy analysis reveals how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore.

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria and plant chloroplasts; the crystal structure of TatC, an integral membrane protein and core component of this complex, is now presented.

KUP transporters facilitate potassium uptake by the co-transport of protons and are key players in potassium homeostasis. Here authors identify the potassium importer KimA from Bacillus subtilis as a new member of the KUP transporter family and show the cryo-EM structure of KimA in an inward-occluded, trans-inhibited conformation.

Lipid droplet biogenesis is orchestrated by the conserved membrane protein Seipin via an unknown mechanism. Here, the authors use structural, biochemical and molecular dynamics simulation approaches to reveal the mechanism of lipid droplet formation by the yeast Seipin Sei1 and its partner Ldb16.

Filamentous phages can assemble into mesoscale structures termed tactoids that protect bacteria in biofilms from antibiotics. Here, the authors dissect the biophysical factors influencing this protection using two model phages, Pf4 and fd.

Live-cell single-molecule imaging reveals a single population of processive septal peptidoglycan synthases moving asynchronously with FtsZ that drive Bacillus subtilis cell constriction in a manner partially dependent upon FtsZ treadmilling.

Cryo-EM structures of human SERINC5 and its fly orthologue, along with extensive functional analyses, reveal the protein architecture and identify regions important for HIV-1 restriction.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Here authors introduce an E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays that are visualized by cryo-electron tomography.

Using single-particle analysis cryogenic electron microscopy, the authors determine the structures of the bacterial flagellum stator complexes from three diverse bacteria.

Diacylglycerol kinase is a small bacterial membrane-bound trimer that catalyses diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution.

Lipopolysaccharide, an essential component of the outer membranes of Gram-negative bacteria, is inserted by LptD–LptE, a protein complex with a unique ‘barrel and plug’ architecture; the structure, molecular dynamics simulations and functional assays of the LptD–LptE complex of Salmonella typhimurium suggest that lipopolysaccharide may pass through the barrel and is then inserted into the outer leaflet of the outer membrane through a lateral opening between two β-strands of LptD.

Lipoproteins are essential components of bacterial membranes. Here the authors present the crystal structures ofPseudomonas aeruginosa and Escherichia coliapolipoprotein N-acyltransferase, which catalyses the final step in the lipoprotein synthesis pathway, and give insights into its mechanism.

A Kv1 channel inhibitor and potential therapeutic lead achieves selectivity by binding both the conserved central cavity and newly identified side pockets, which provide the key determinants for channel specificity.

Bacterial cell wall components are assembled in a transmembrane cycle that involves the membrane integral pyrophosphorylase, BacA. Here the authors solve the crystal structure of BacA which shows an interdigitated inverted topology repeat that hints towards a flippase function for BacA.

Membrane lipids such as cardiolipin act as molecular glue to preserve the oligomeric states of membrane proteins with low oligomeric stability.

Protein-lipid interactions can mediate the function of membrane proteins. Here coarse-grained molecular dynamics free energy perturbation simulations yield binding free energies for phosphoinositide lipids to hKir channels and mutants, in agreement with electrophysiological measurements.

Gain-of-function mutations in the genes encoding ATP-sensitive potassium channel (K_ATP channel) subunits cause neonatal diabetes mellitus. This Review discusses the mechanism of action of mutations that lead to neonatal diabetes mellitus and briefly reviews work on the management of this disease.

C. Keith Cassidy et al. use cryo-electron tomography and subtomogram averaging to examine the structure of the core-signalling units of E. coli chemotaxis arrays at subnanometer resolution. They find multiple distinct conformations of the critical CheA kinase domain, offering new insights into CheA signalling.