MP

Structure of tetrameric forms of the serotonin-gated 5-HT3_A receptor ion channel. 

Introini B, Cui W, Chu X, Zhang Y, Alves AC, Eckhardt-Strelau L, Golusik S, Tol M, Vogel H, Yuan S, Kudryashev M.

EMBO J. 2024 Sep 4. 

doi: 10.1038/s44318-024-00191-5. Epub ahead of print. 

PMID: 39232129.

Multimeric membrane proteins are produced in the endoplasmic reticulum and transported to their target membranes which, for ion channels, is typically the plasma membrane. Despite the availability of many fully assembled channel structures, our understanding of assembly intermediates, multimer assembly mechanisms, and potential functions of non-standard assemblies is limited. We demonstrate that the pentameric ligand-gated serotonin 5-HT3A receptor (5-HT3AR) can assemble to tetrameric forms and report the structures of the tetramers in plasma membranes of cell-derived microvesicles and in membrane memetics using cryo-electron microscopy and tomography. The tetrameric structures have near-symmetric transmembrane domains, and asymmetric extracellular domains, and can bind serotonin molecules. Computer simulations, based on our cryo-EM structures, were used to decipher the assembly pathway of pentameric 5-HT3R and suggest a potential functional role for the tetrameric receptors. 

Structures of the Mycobacterium tuberculosis efflux pump EfpA reveal the mechanisms of transport and inhibition. 

Wang S, Wang K, Song K, Lai ZW, Li P, Li D, Sun Y, Mei Y, Xu C, Liao M.

Nat Commun. 2024 Sep 4;15(1):7710. 

doi: 10.1038/s41467-024-51948-9. 

PMID: 39231991.

As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs. 

Dynamic basis of lipopolysaccharide export by LptB₂FGC

Marina Dajka, Tobias Rath, Nina Morgner, Benesh Joseph

bioRxiv 2024.05.20.594984; doi: https://doi.org

10.1101/2024.05.20.594984 

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB₂FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB₂FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB₂FG and LptB₂FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB₂FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

Transport of herbicides by PIN-FORMED auxin transporters

Lukas Schulz, Kien Lam Ung, Sarah Koutnik-Abele, David L Stokes, Bjørn Panyella Pedersen, Ulrich Z. Hammes

bioRxiv 2024.08.29.610046; 

doi: https://doi.org/10.1101/2024.08.29.610046 

Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane and we characterize the molecular determinants of this process using a variety of different substrates as well as protein mutagenesis to control substrate specificity. Finally, we present Cryo-EM structures of Arabidopsis thaliana PIN8 with 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-chlorophenoxyacetic acid (4-CPA) bound. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with substrate binding and transport across the membrane. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results enable development of novel synthetic auxins and for guiding precision breeding of herbicide resistant crop plants.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis. 

Babii S, Li W, Yang L, Grzegorzewicz AE, Jackson M, Gumbart JC, Zgurskaya HI.

mBio. 2024 Aug 30:e0218324. 

doi: 10.1128/mbio.02183-24. Epub ahead of print. 

PMID: 39212407.

Infections caused by Mycobacterium spp. are very challenging to treat and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria drug efflux and the low permeability barrier of their outer membrane. Here we focus on MmpL3 from Mycobacterium tuberculosis an essential inner membrane transporter of the resistance–nodulation–division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro inside macrophages and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3 coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.

NH₃/NH₄⁺ allosterically activates SLC4A11 by causing an acidic shift in the intracellular pK that governs H⁺(OH^–) conductance. 

Pasternack RA, Quade BN, Marshall A, Parker MD.

Front Physiol. 2024 Aug 14;15:1440720. 

doi: 10.3389/fphys.2024.1440720. 

PMID: 39206384.

SLC4A11 is the most abundant membrane transport protein in corneal endothelial cells. Its functional presence is necessary to support the endothelial fluid pump that draws fluid from the corneal stroma, preventing corneal edema. Several molecular actions have been proposed for SLC4A11 including H₂O transport and cell adhesion. One of the most reproduced actions that SLC4A11 mediates is a H⁺ (or OH^–) conductance that is enhanced in the presence of NH₄Cl. The mechanism by which this occurs is controversial with some providing evidence in favor of NH₃-H⁺ cotransport and others providing evidence for uncoupled H⁺ transport that is indirectly stimulated by the effects of NH₄Cl upon intracellular pH and membrane potential. In the present study we provide new evidence and revisit previous studies, to support a model in which NH₄Cl causes direct allosteric activation of SLC4A11 by means of an acidic shift in the intracellular pK (pK_i) that governs the relationship between intracellular pH (pH_i) and SLC4A11 H⁺-conductance. These findings have important implications for the assignment of a physiological role for SLC4A11. 

Molecular recognition of an odorant by the murine trace amine-associated receptor TAAR7f. 

Gusach A, Lee Y, Khoshgrudi AN, Mukhaleva E, Ma N, Koers EJ, Chen Q, Edwards PC, Huang F, Kim J, Mancia F, Veprintsev DB, Vaidehi N, Weyand SN, Tate CG.

Nat Commun. 2024 Aug 30;15(1):7555. 

doi: 10.1038/s41467-024-51793-w. 

PMID: 39215004.

There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin, dopamine and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein G_s and bound to the odorant N,N-dimethylcyclohexylamine (DMCHA) to an overall resolution of 2.9 Å. DMCHA is bound in a hydrophobic orthosteric binding site primarily through van der Waals interactions and a strong charge-charge interaction between the tertiary amine of the ligand and an aspartic acid residue. This site is distinct and non-overlapping with the binding site for the odorant propionate in the odorant receptor OR51E2. The structure, in combination with mutagenesis data and molecular dynamics simulations suggests that the activation of the receptor follows a similar pathway to that of the β-adrenoceptors, with the significant difference that DMCHA interacts directly with one of the main activation microswitch residues, Trp^6.48. 

Interdomain-linkers control conformational transitions in the SLC23 elevator transporter UraA. 

Kuhn BT, Zöller J, Zimmermann I, Gemeinhardt T, Özkul DH, Langer JD, Seeger MA, Geertsma ER.

Nat Commun. 2024 Aug 30;15(1):7518. 

doi: 10.1038/s41467-024-51814-8. 

PMID: 39209842.

Uptake of nucleobases and ascorbate is an essential process in all living organisms mediated by SLC23 transport proteins. These transmembrane carriers operate via the elevator alternating-access mechanism, and are composed of two rigid domains whose relative motion drives transport. The lack of large conformational changes within these domains suggests that the interdomain-linkers act as flexible tethers. Here, we show that interdomain-linkers are not mere tethers, but have a key regulatory role in dictating the conformational space of the transporter and defining the rotation axis of the mobile transport domain. By resolving a wide inward-open conformation of the SLC23 elevator transporter UraA and combining biochemical studies using a synthetic nanobody as conformational probe with hydrogen-deuterium exchange mass spectrometry, we demonstrate that interdomain-linkers control the function of transport proteins by influencing substrate affinity and transport rate. These findings open the possibility to allosterically modulate the activity of elevator proteins by targeting their linkers. 

Outer membrane protein assembly mediated by BAM-SurA complexes. 

Fenn KL, Horne JE, Crossley JA, Böhringer N, Horne RJ, Schäberle TF, Calabrese AN, Radford SE, Ranson NA.

Nat Commun. 2024 Sep 1;15(1):7612. 

doi: 10.1038/s41467-024-51358-x. 

PMID: 39218969.

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis. 

Crossing the membrane – what does it take to flip a phospholipid Structural and biochemical advances on P4-ATPase flippases. 

Sai KV, Lee JY.

J Biol Chem. 2024 Sep 2:107738. 

doi: 10.1016/j.jbc.2024.107738. Epub ahead of print. 

PMID: 39233230.

Membrane asymmetry is critical for maintenance of several different processes such as cell signalling, apoptosis, and vesicular transport in various eukaryotic systems. Flippases of the P4-ATPase family are associated with flipping phospholipids from the luminal or exoplasmic leaflet to the cytosolic leaflet. P4-ATPases belong to the P-type ATPase family, which are activated by phosphorylation and couple ATPase activity to substrate translocation. These proteins possess a transmembrane domain responsible for substrate transport, while the cytosolic machinery perform the necessary ATP hydrolysis for this process. Several high-resolution structures of human or yeast P4-ATPases have recently been resolved, but a comprehensive overview of the changes for reaction cycle in different members was crucial for future research. In this review, we have compiled available data reflecting the reaction cycle-associated changes in conformation of P4-ATPases. Together, this will provide an improved understanding of the similarities and differences between these members, which will drive further structural, functional and computational studies to understand the mechanisms of these flippases. 

Membranes

In-cell architecture of the mitochondrial respiratory chain

Florent Waltz, Ricardo D. Righetto, Ron Kelley, Xianjun Zhang, Martin Obr, Sagar Khavnekar, Abhay Kotecha, Benjamin D. Engel

bioRxiv 2024.09.03.610704; doi: https://doi.org/10.1101/2024.09.03.610704 

Mitochondria produce energy through oxidative phosphorylation, carried out by five membrane-bound complexes collectively known as the respiratory chain. These complexes work in concert to transfer electrons and pump protons, leading to ATP regeneration. The precise organization of these complexes in native cells is debated, notably their assembly into higher-order supercomplexes called respirasomes. Here, we use in situ cryo-electron tomography to visualize the native structures and organization of several major mitochondrial complexes inside Chlamydomonas reinhardtii cells. ATP synthases and respiratory complexes are segregated into curved and flat crista membrane domains, respectively. Respiratory complexes I, III, and IV assemble into a single type of respirasome, from which we determined a native 5 Å-resolution structure showing the binding of electron carrier cytochrome c. Combined with single-particle cryo-electron microscopy reconstruction at 2.4 Å resolution, we assemble a detailed model of how the respiratory complexes interact with each other inside native mitochondria.

Caveolin assemblies displace one bilayer leaflet to organize and bend membranes

Milka Doktorova, Sebastian Daum, Jan Ebenhan, Sarah Neudorf, Bing Han, Satyan Sharma, Peter Kasson, Kandice R. Levental, Kirsten Bacia, Anne K. Kenworthy, Ilya Levental

bioRxiv 2024.08.28.610209; 

doi: https://doi.org/10.1101/2024.08.28.610209 

Caveolin is a monotopic integral membrane protein, widely expressed in metazoa and responsible for constructing enigmatic membrane invaginations known as caveolae. Recently, the high-resolution structure of a purified human caveolin assembly, the CAV1-8S complex, revealed a unique organization of 11 protomers arranged in a tightly packed, radially symmetric spiral disc. One face and the outer rim of this disc are highly hydrophobic, suggesting that the complex incorporates into membranes by displacing hundreds of lipids from one leaflet. The feasibility of this unique molecular architecture and its biophysical and functional consequences are currently unknown. Using Langmuir film balance measurements, we find that CAV1-8S is highly surface active and intercalates into lipid monolayers. Molecular simulations of biomimetic bilayers support this ‘leaflet replacement’ model and reveal that while CAV1-8S effectively displaces phospholipids from one bilayer leaflet, it accumulates 40−70 cholesterol molecules into a disordered monolayer between the complex and its distal lipid leaflet. We find that CAV1-8S preferentially associates with positively curved membrane surfaces due to its influence on the conformations of distal leaflet lipids, and that these effects laterally sort lipids of the distal leaflet. Large-scale simulations of multiple caveolin assemblies confirmed their association with large, positively curved membrane morphologies, consistent with the shape of caveolae. Further, association with curved membranes regulates the exposure of caveolin residues implicated in protein-protein interactions. Altogether, the unique structure of CAV1-8S imparts unusual modes of membrane interaction with implications for membrane organization, morphology, and physiology.

Self-diffusion is temperature independent on active membranes. 

Varma SG, Mitra A, Sarkar S.

Phys Chem Chem Phys. 2024 Sep 11;26(35):23348-23362. 

doi: 10.1039/d4cp02470b. 

PMID: 39211961.

Molecular transport maintains cellular structures and functions. For example, lipid and protein diffusion sculpts the dynamic shapes and structures on the cell membrane that perform essential cellular functions, such as cell signaling. Temperature variations in thermal equilibrium rapidly change molecular transport properties. The coefficient of lipid self-diffusion increases exponentially with temperature in thermal equilibrium, for example. Hence, maintaining cellular homeostasis through molecular transport is hard in thermal equilibrium in the noisy cellular environment, where temperatures can fluctuate widely due to local heat generation. In this paper, using both molecular and lattice-based modeling of membrane transport, we show that the presence of active transport originating from the cell’s cytoskeleton can make the self-diffusion of the molecules on the membrane robust to temperature fluctuations. The resultant temperature-independence of self-diffusion keeps the precision of cellular signaling invariant over a broad range of ambient temperatures, allowing cells to make robust decisions. We have also found that the Kawasaki algorithm, the widely used model of lipid transport on lattices, predicts incorrect temperature dependence of lipid self-diffusion in equilibrium. We propose a new algorithm that correctly captures the equilibrium properties of lipid self-diffusion and reproduces experimental observations. 

Molecules

Lipid-polymer nanoparticles to probe the native-like environment of intramembrane rhomboid protease GlpG and its activity. 

Sawczyc H, Tatsuta T, Öster C, Kosteletos S, Lange S, Bohg C, Langer T, Lange A.

Nat Commun. 2024 Aug 30;15(1):7533. 

doi: 10.1038/s41467-024-51989-0. 

PMID: 39215029.

Polymers can facilitate detergent-free extraction of membrane proteins into nanodiscs (e.g., SMALPs, DIBMALPs), incorporating both integral membrane proteins as well as co-extracted native membrane lipids. Lipid-only SMALPs and DIBMALPs have been shown to possess a unique property; the ability to exchange lipids through ‘collisional lipid mixing’. Here we expand upon this mixing to include protein-containing DIBMALPs, using the rhomboid protease GlpG. Through lipidomic analysis before and after incubation with DMPC or POPC DIBMALPs, we show that lipids are rapidly exchanged between protein and lipid-only DIBMALPs, and can be used to identify bound or associated lipids through ‘washing-in’ exogenous lipids. Additionally, through the requirement of rhomboid proteases to cleave intramembrane substrates, we show that this mixing can be performed for two protein-containing DIBMALP populations, assessing the native function of intramembrane proteolysis and demonstrating that this mixing has no deleterious effects on protein stability or structure. 

Recent advance in Artificial Anion Channels and Their Selectivity. 

Ren B, Sun Y, Xin P.

Chempluschem. 2024 Aug 30:e202400466. 

doi: 10.1002/cplu.202400466. Epub ahead of print. 

PMID: 39212532.

Nature performs critical physiological functions using a series of structurally and functionally diverse membrane proteins embedded in cell membranes, in which native ion protein channels modify the electrical potential inside and outside the cell membrane through charged ion movements. Consequently, the cell responds to external stimuli, playing an essential role in various life activities, such as nerve excitation conduction, neurotransmitter release, muscle movement, and control of cell differentiation. Supramolecular artificial channels, which mimic native protein channels in structure and function, adopt unimolecular or self-assembled structures, such as crown ethers, cyclodextrins, cucurbiturils, column arenes, cyclic peptide nanotubes, and metal-organic artificial channels, in channel construction strategies. Owing to the various driving forces involved, artificial synthetic ion channels can be divided into artificial cation and anion channels in terms of ion selectivity. Cation selectivity usually originates from ion coordination, whereas anion selectivity is related to hydrogen bonding, ion pairing, and anion-dipole interactions. Several studies have been conducted on artificial cation channels, and several reviews have summarized them in detail; however, the research on anions is still in the initial stages, and related reviews have rarely been reported. Hence, this article primarily focuses on the recent research on anion channels. 

Photo-induced polymerization of styrene-maleic acid copolymers for the extraction of membrane proteins

V Monjal, P Guillet, A Moreno, M Soulié, G Durand

Journal of Polymer Science, 2024

doi: 10.1002/pol.20240295

http://wileyonlinelibrary.com/journal/pol

We report herein the photoinduced electron/energy transfer–reversible addition-fragmentation chain transfer (PET-RAFT) polymerization of styrene-maleic anhydride (SMAnh) using the photocatalyst, zinc tetraphenylporphir-ine, under a white cool LED light. PET-RAFT is an easy and convenient poly-merization method that does not require deoxygenation unlike other radical polymerizations. Various parameters, for example, the amount of styrene in the feed or the solvent, and their influence on the polymerization were studied. After hydrolysis of the anhydride moieties, the resulting styrene-maleic acid (SMA) copolymers obtained by PET-RAFT copolymerization were evaluated for their solubilization efficiency of three different membrane proteins, BmrA and AcrB, overexpressed in Escherichia coli, and A 2A R, expressed in insect cells (Sf9). The different copolymers provided similar solubilization rates to the commercially available SMA; however, a highly improved migration behavior on sodium dodecyl-sulfate polyacrylamide gel electrophoresis was observed which could, facilitate downstream analyses. Overall, we demonstrated that PET-RAFT is a versatile oxygen tolerant polymerization technique to yield SMA, a suitable polymer for biochemical applications.

Methods

Impact of Cellular Crowding on Protein Structural Dynamics Investigated by EPR Spectroscopy. 

Pierro A, Bonucci A, Magalon A, Belle V, Mileo E.

Chem Rev. 2024 Sep 11;124(17):9873-9898. 

doi: 10.1021/acs.chemrev.3c00951. Epub 2024 Aug 30. 

PMID: 39213496.

Genetic Code Expansion for Mechanistic Studies in Ion Channels: An (Un)natural Union of Chemistry and Biology. 

Infield DT, Schene ME, Galpin JD, Ahern CA.

Chem Rev. 2024 Aug 29. 

doi: 10.1021/acs.chemrev.4c00306. Epub ahead of print. 

PMID: 39207057.

Ion channels play central roles in biology and human health by catalyzing the transmembrane flow of electrical charge. These proteins are ideal targets for genetic code expansion (GCE) methods because it is feasible to measure ion channel activity from miniscule amounts of protein and to analyze the resulting data via rigorous, established biophysical methods. In an ideal scenario, the encoding of synthetic, noncanonical amino acids via GCE allows the experimenter to ask questions inaccessible to traditional methods. For this reason, GCE has been successfully applied to a variety of ligand- and voltage-gated channels wherein extensive structural, functional, and pharmacological data exist. Here, we provide a comprehensive summary of GCE as applied to ion channels. We begin with an overview of the methods used to encode noncanonical amino acids in channels and then describe mechanistic studies wherein GCE was used for photochemistry (cross-linking; caged amino acids) and atomic mutagenesis (isosteric manipulation of charge and aromaticity; backbone mutation). Lastly, we cover recent advances in the encoding of fluorescent amino acids for the real-time study of protein conformational dynamics. 

Attosecond electron microscopy and diffraction. 

Hui D, Alqattan H, Sennary M, Golubev NV, Hassan MT.

Sci Adv. 2024 Aug 23;10(34):eadp5805. 

doi: 10.1126/sciadv.adp5805. Epub 2024 Aug 21. 

PMID: 39167650.

Advances in attosecond spectroscopy have enabled tracing and controlling the electron motion dynamics in matter, although they have yielded insufficient information about the electron dynamic in the space domain. Hence, ultrafast electron and x-ray imaging tools have been developed to image the ultrafast dynamics of matter in real time and space. The cutting-edge temporal resolution of these imaging tools is on the order of a few tens to a hundred femtoseconds, limiting imaging to the atomic dynamics and leaving electron motion imaging out of reach. Here, we obtained the attosecond temporal resolution in the transmission electron microscope, which we coined “attomicroscopy.” We demonstrated this resolution by the attosecond diffraction measurements of the field-driven electron dynamics in graphene. This attosecond imaging tool would provide more insights into electron motion and directly connect it to the structural dynamics of matter in real-time and space domains, opening the door for long-anticipated real-life attosecond science applications in quantum physics, chemistry, and biology. 

Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM. 

Li C, Willegems K, Uchański T, Pardon E, Steyaert J, Efremov RG.

J Biol Chem. 2024 Sep 2:107734. 

doi: 10.1016/j.jbc.2024.107734. Epub ahead of print. 

PMID: 39233227.

Ryanodine receptors (RyRs) are large Ca²⁺ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within four hours on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited. 

Microbio

Efflux of TolC protein to different antimicrobials can be replaced by other outer membrane proteins with similar β-barrel structures in extraintestinal pathogenic Escherichia coli. 

Bao X, Yang C, Li T, Wang Y, Cui A, Meng X, Huang Q, Li S.

J Appl Microbiol. 2024 Sep 2;135(9):lxae214. 

doi: 10.1093/jambio/lxae214. 

PMID: 39217099.

OMPs can replace the TolC protein to play the efflux role in pumping out the drugs from the periplasm to the extracellular space with the help of proteins AcrA and AcrB. ???

Structural and functional diversity of Resistance-Nodulation-Division (RND) efflux pump transporters with implications for antimicrobial resistance. 

Kavanaugh LG, Dey D, Shafer WM, Conn GL.

Microbiol Mol Biol Rev. 2024 Sep 5:e0008923. 

doi: 10.1128/mmbr.00089-23. Epub ahead of print. 

PMID: 39235227.

The discovery of bacterial efflux pumps significantly advanced our understanding of how bacteria can resist cytotoxic compounds that they encounter. Within the structurally and functionally distinct families of efflux pumps, those of the Resistance-Nodulation-Division (RND) superfamily are noteworthy for their ability to reduce the intracellular concentration of structurally diverse antimicrobials. RND systems are possessed by many Gram-negative bacteria, including those causing serious human disease, and frequently contribute to resistance to multiple antibiotics. Herein, we review the current literature on the structure-function relationships of representative transporter proteins of tripartite RND efflux pumps of clinically important pathogens. We emphasize their contribution to bacterial resistance to clinically used antibiotics, host defense antimicrobials and other biocides, as well as highlighting structural similarities and differences among efflux transporters that help bacteria survive in the face of antimicrobials. Furthermore, we discuss technical advances that have facilitated and advanced efflux pump research and suggest future areas of investigation that will advance antimicrobial development efforts. 

Miscellaneous

Massive Attack’s science-led drive to lower music’s carbon footprint. 

Hedley E.

Nature. 2024 Sep;633(8028):241-243. 

doi: 10.1038/d41586-024-02835-2. 

PMID: 39232153.

The heat is on: reduced detection of floral scents after heatwaves in bumblebees. 

Nooten SS, Korten H, Schmitt T, Kárpáti Z.

High temperatures seem to disrupt bumblebees’ ability to smell a sense they rely on to feed and pollinate. Exposing the antennae of two species of bumblebee (Bombus pascuorum and Bombus terrestris) to 40 oC heat for just 3 hours made them significantly less responsive to scent molecules a change that persisted a day later. “The results are pretty clear: There is an effect of heat waves on bumble bee physiology” says ecologist Coline Jaworski.

HIV: how close are we to a vaccine – or a cure? 

Kwon D.

Nature. 2024 Sep 2. 

doi: 10.1038/d41586-024-02840-5. Epub ahead of print. 

PMID: 39223275.

Antiretroviral therapy has saved and changed lives by preventing the HIV virus from replicating inside the body. And there are a handful of people who have been cured with disease-resistant stem-cells as part of a painful and risky bone-marrow transplant that they needed anyway to treat cancer. But scientists are on the hunt for more long-term scalable solutions. These include gene therapies that prevent the virus from entering cells or target the virus itself. Other efforts try to control or eliminate the latent pool of HIV-infected cells that do not produce viral particles. And some injectable antivirals approved as long-acting HIV treatments have also proved to be effective at preventing infection. The field is also making steady progress towards a vaccine but there’s still a long way to go.

An extraordinary larval-like teleost fish from the Eocene of Bolca

Donald Davesne, Giorgio Carnevale

bioRxiv 2024.08.19.608581; 

doi: https://doi.org/10.1101/2024.08.19.608581 

Freaky fish might need a new name !

The biology of smell is a mystery – AI is helping to solve it. 

Smith K.

Nature. 2024 Sep;633(8028):26-29. 

doi: 10.1038/d41586-024-02833-4. 

PMID: 39227712.