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Protein tertiary structure mimetics are valuable tools to target large protein–protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein–protein interaction through target dimerization.

Antibiotic resistance is a growing global health concern. Colistin is one of the last-resort antibiotics that treats multidrug-resistant (MDR) Gram-negative bacterial infection. However, bacteria resistant to colistin have become increasingly prevalent. Using a bacterial whole-cell screen of a fragment-based library, one compound was discovered to resensitize MDR

The structural and characteristic features of HIV-1 broadly neutralizing antibodies (bnAbs) from chronically infected pediatric donors are currently unknown. Herein, we characterized a heavy chain matured HIV-1 bnAb 44m, identified from a pediatric elite-neutralizer. Interestingly, in comparison to its wild-type AIIMS-P01 bnAb, 44m exhibited moderately higher level of somatic hypermutations (SHM) of 15.2%. 44m neutralized 79% of HIV-1 heterologous viruses tested, with a geometric mean IC50titer of 0.36 μg/ml. The cryoEM structure of 44m Fab in complex with fully-cleaved glycosylated native-like BG505.SOSIP envelope trimer at 4.4 Å resolution revealed that 44m targets the V3-glycan N332-supersite and GDIR motif to neutralize HIV-1 with improved potency and breadth, plausibly attributed by a matured heavy chain as compared to that of wild-type AIIMS-P01 bnAb. This study improves our understanding on pediatric HIV-1 bnAbs and structural basis of broad HIV-1 neutralization by 44m may be useful blueprint for vaccine design in future.

DNA-binding protein under starvation (Dps), is a miniature ferritin complex which plays a vital role in protecting bacterial DNA during starvation for maintaining the integrity of bacteria from hostile conditions.Mycobacterium smegmatisis one such bacteria that express MsDps2, which binds DNA to protect it under oxidative and nutritional stress conditions. Several approaches, including cryo-electron tomography (Cryo-ET), were implemented to identify the structure of the Dps protein that is bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to be able to identify the DNA binding residues. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps protein. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA and identify that the region close to N-terminal confers the DNA binding property. Based on cryo-EM data, we performed mutations of several arginine residues proximal to DNA binding region, which dramatically reduced the MsDps2-DNA interaction. In addition, we demonstrated the proposed model for DNA compaction during lattice formation. We also pinpointed arginine residues, which are responsible for DNA binding in lattice arrangement of MsDps2. We performed single-molecule imaging experiments of MsDps2-DNA interactions that corroborate well with our structural studies.

We present new 3-mm continuum and molecular lines observations from the ATOMS survey towards the massive protostellar clump, MM1, located in the filamentary infrared dark cloud (IRDC), G034.43+00.24 (G34). The lines observed are the tracers of either dense gas (e.g. HCO+/H13CO+ J = 1-0) or outflows (e.g. CS J = 2-1). The most complete picture to date of seven cores in MM1 is revealed by dust continuum emission. These cores are found to be gravitationally bound, with virial parameter, $\alpha_{vir}, Comment: 14 pages with 6 figures, and in press

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency.HighlightsTwo potent human mAbs obtained from a single donor differ binding to Omicron spikesPattern of binding and conformation of these mAbs bound to full length spike differsAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms.Cryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs.

Mycobacterium tuberculosis utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. ESX-1 is one of the essential and well-studied secretion systems which transport various virulence factors, including EspB. EspB, a ~36 kDa secreted substrate, has been implicated to play vital role in protecting the bacteria from hostile environment within the host cell phagosome. It is also involved in bacterial pathogenesis and has been shown to bind phospholipids. Recently, two cryo-EM structures of EspB full-length and the secreted isoforms were resolved. Despite the availability of multiple high-resolution structures of EspB, the physiological relevance and mechanism of virulence of this secreted substrate remains poorly characterized. In this current work, we implemented cryo-EM-based structural studies, including various functional assays, TEM imaging, and biophysical approach to demonstrate the interaction of EspB with lipids and bio-membrane. Our findings also indicated that EspB may play a crucial role in binding to and rupturing host mitochondrial membrane. Through cryo-EM studies we were able to show the possible membrane-binding region of EspB. Our study sheds light on host-pathogen interactions and bacterial pathogenesis mediated by EspB.

γ-hemolysin (γ-HL) is a hemolytic and leukotoxic bicomponent β-pore-forming toxin (β-PFT), a potent virulence factor from Staphylococcus aureus Newman strain. In this study, we performed single particle cryo-EM of γ-HL in a lipid environment. We observed clustering and square lattice packing of octameric HlgAB pores upon membrane bilayer, and an octahedral superassembly of octameric pore complexes, that we resolved at resolution 3.5 Å. Our atomic model further demonstrated the key residues involved in hydrophobic zipping between the rim domains of adjacent octameric pore complexes, thus providing first evidence of additional structural stability in PFTs upon membrane lysis. We also observed lipid densities at the octahedral and octameric interfaces, providing critical insights into the lipid-binding residues involved for both HlgA and HlgB components. Furthermore, the hitherto elusive N-terminal region of HlgA has also been resolved in our cryo-EM map and an overall mechanism of pore formation for bicomponent β-PFTs is proposed.

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.

Methionine and cysteine metabolisms are important for the survival and pathogenesis ofMycobacterium tuberculosis(Mtb). The transsulfuration pathway converts methionine to cysteine and represents an important link between antioxidant and methylation metabolism in diverse organisms. Using a combination of biochemistry and cryo–electron microscopy, we characterized the first enzyme of the transsulfuration pathway, cystathionine β-synthase (MtbCbs) inMtb. We demonstrated thatMtbCbs is a heme-less, pyridoxal-5′-phosphate–containing enzyme, allosterically activated byS-adenosylmethionine (SAM). The atomic model ofMtbCbs in its native and SAM-bound conformations revealed a unique mode of SAM-dependent allosteric activation. Further, SAM stabilizedMtbCbs by sterically occluding proteasomal degradation, which was crucial for supporting methionine and redox metabolism inMtb. Genetic deficiency ofMtbCbs reducedMtbsurvival upon homocysteine overload in vitro, inside macrophages, and in mice coinfected with HIV. Thus, theMtbCbs-SAM axis constitutes an important mechanism of coordinating sulfur metabolism inMtb.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Multidrug-resistant (MDR) Gram-negative bacteria are an urgent and rapidly spreading threat to human health with limited treatment options. Previously, we discovered a novel [1,2,5]oxadiazolo[3,4-b]pyrazine-containing compound (1) that selectively re-sensitized a variety of MDR Gram-negative bacteria to colistin, one of the last-resort antibiotic. Herein, we report the structure–activity relationship studies of compound 1 that led to the discovery of several more potent and/or less toxic resistance-modifying agents (RMAs). Further evaluation of these RMAs showed that they were effective in a wide range of MDR bacteria. These results demonstrated these compounds as a novel class of RMAs and may be further developed as therapeutic agents.

β-barrel pore-forming toxins perforate cell membranes by forming oligomeric β-barrel pores. The most crucial step is the membrane-insertion of the pore-forming motifs that create the transmembrane β-barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore-forming motifs during β-barrel pore-formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the β-barrel pore-forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore-forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane-bound toxin protomers, and blocks subsequent steps of pore-formation. Our study suggests that the presence of Y321 in the hinge region of the pore-forming motif is crucial for the toxin molecule to sense membrane-binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore-formation process. Such a regulatory mechanism of pore-formation by V. cholerae cytolysin has not been documented earlier in the structurally related β-barrel pore-forming toxins.

We have conducted a line survey towards Orion KL using the Q-band receiver of Tianma 65 m radio telescope (TMRT), covering 34.8--50 GHz with a velocity resolution between 0.79 km s$^{-1}$ and 0.55 km s$^{-1}$ respectively. The observations reach a sensitivity on the level of 1-8 mK, proving that the TMRT is sensitive for conducting deep line surveys. In total, 597 Gaussian features are extracted. Among them, 177 radio recombination lines (RRLs) are identified, including 126, 40 and 11 RRLs of hydrogen, helium and carbon, with a maximum $\Delta n$ of 16, 7, and 3, respectively. The carbon RRLs are confirmed to originate from photodissociation regions with a $V_{\rm LSR}\sim$9 km s$^{-1}$. In addition, 371 molecular transitions of 53 molecular species are identified. Twenty-one molecular species of this survey were not firmly detected in the Q band by Rizzo et al. (2017), including species such as H$_2$CS, HCOOH, C$_2$H$_5$OH, H$_2^{13}$CO, H$_2$CCO, CH$_3$CHO, CH$_2$OCH$_2$, HCN $v_2=1$, and CH$_3$OCHO $v_t=1$. In particular, the vibrationally excited states of ethyl cyanide (C$_2$H$_5$CN $v$13/$v$21) are for the first time firmly detected in the Q band. NH$_3$ (15,15) and (16,16) are identified, and they are so far the highest transitions of the NH$_3$ inversion lines detected towards Orion KL. All the identified lines can be reproduced by a radiative transfer model., Comment: 51 pages, 18 figures, accepted by ApJS

Rapid emergence of the SARS-CoV-2 variants has dampened the protective efficacy of existing authorized vaccines. Nanoparticle platforms offer a means to improve vaccine immunogenicity by presenting multiple copies of desired antigens in a repetitive manner which closely mimics natural infection. We have applied nanoparticle display combined with the SpyTag–SpyCatcher system to design encapsulin–mRBD, a nanoparticle vaccine displaying 180 copies of the monomeric SARS-CoV-2 spike receptor-binding domain (RBD). Here we show that encapsulin–mRBD is strongly antigenic and thermotolerant for long durations. After two immunizations, squalene-in-water emulsion (SWE)-adjuvanted encapsulin–mRBD in mice induces potent and comparable neutralizing antibody titers of 105 against wild-type (B.1), alpha, beta, and delta variants of concern. Sera also neutralizes the recent Omicron with appreciable neutralization titers, and significant neutralization is observed even after a single immunization.

Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small β-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer–embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 °C and were stable to long term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼ three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Thermostable direct hemolysin (TDH) of Vibrio parahemolyticus is a membrane-damaging pore-forming toxin with potent cytolytic/cytotoxic activity. TDH exists as a tetramer consisting of protomers with a core β-sandwich domain, flanked by an 11-amino acid long N-terminal region (NTR). This NTR could not be modeled in the previously determined crystal structure of TDH. Moreover, the functional implication of NTR for the membrane-damaging action of TDH remains unknown. In the present study, we have explored the implications of NTR for the structure-function mechanism of TDH. Our data show that the presence of NTR modulates the physicochemical property of TDH in terms of augmenting the amyloidogenic propensity of the protein. Deletion of NTR compromises the binding of TDH toward target cell membranes and drastically affects the membrane-damaging cytolytic/cytotoxic activity of the toxin. Mutations of aromatic/hydrophobic residues within NTR also confer compromised cell-killing activity. Moreover, covalent trapping of NTR, via an engineered disulfide bond, against the core β-sandwich domain also abrogates the cytolytic/cytotoxic activity of TDH. This observation suggests that an unrestrained configuration of NTR is crucial for the membrane-damaging action of TDH. On the basis of our study, we propose a model explaining the role of NTR in the membrane-damaging function of TDH.

Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Prestellar cores are self-gravitating dense and cold structures within molecular clouds where future stars are born. They are expected, at the stage of transitioning to the protostellar phase, to harbor centrally concentrated dense (sub)structures that will seed the formation of a new star or the binary/multiple stellar systems. Characterizing this critical stage of evolution is key to our understanding of star formation. In this work, we report the detection of high-density (sub)structures on the thousand-astronomical-unit (au) scale in a sample of dense prestellar cores. Through our recent ALMA observations toward the Orion Planck Galactic Cold Clumps, we have found five extremely dense prestellar cores, which have centrally concentrated regions of ∼2000 au in size, and several 107 cm-3 in average density. Masses of these centrally dense regions are in the range of 0.30 to 6.89M⊙. For the first time, our higher resolution observations (0.8' ∼ 320 au) further reveal that one of the cores shows clear signatures of fragmentation; such individual substructures/fragments have sizes of 800-1700 au, masses of 0.08 to 0.84M⊙, densities of 2 - 8 × 107 cm-3, and separations of ∼1200 au. The substructures are massive enough (≳0.1M⊙) to form young stellar objects and are likely examples of the earliest stage of stellar embryos that can lead to widely (∼1200 au) separated multiple systems., L. acknowledges the support from the international partnership program of the Chinese Academy of Sciences through grant No.114231KYSB20200009, National Natural Science Foundation of China (NSFC) through grant NSFC No.12073061, and Shanghai Pujiang Program 20PJ1415500. N.H. acknowledges MoST 108-2112-M-001-017 and MoST 109-2112-M-001-023 grants. G.G. acknowledges support from ANID project AFB 170002. L.B. acknowledges support from ANID project AFB-170002. S.L.Q. is supported by the National Natural Science Foundation of China under grant No. U1631237. D.J. is supported by NRC Canada and by an NSERC Discovery Grant. V.M.P. acknowledges support by the Spanish MINECO under project AYA2017-88754-P, and financial support from the State Agency for Research of the Spanish Ministry of Science and Innovation through the “Unit of Excellence María de Maeztu 2020-2023” award to the Institute of Cosmos Sciences (CEX2019-000918-M). C.W.L. is supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2019R1A2C1010851). A.S. acknowledges financial support from the NSF through grant AST-1715876. The research was carried out in part at the Jet Propulsion Laboratory, which is operated for NASA by the California Institute of Technology. D. L. acknowledges support from NSFC No. 11911530226 and 11725313. K.T. was supported by JSPS KAKENHI grant No. 20H05645. J.H. thanks the NSFC grant No. 11873086 and Yunnan Province of China (No. 2017HC018).This work is sponsored (in part) by the Chinese Academy of Sciences (CAS), through a grant to the CAS South America Center for Astronomy (CASSACA) in Santiago, Chile.

Gas at high Galactic latitude is a relatively little-noticed component of the interstellar medium. In an effort to address this, forty-one Planck Galactic Cold Clumps at high Galactic latitude (HGal; $|b|>25^{\circ}$) were observed in $^{12}$CO, $^{13}$CO and C$^{18}$O J=1-0 lines, using the Purple Mountain Observatory 13.7-m telescope. $^{12}$CO (1-0) and $^{13}$CO (1-0) emission was detected in all clumps while C$^{18}$O (1-0) emission was only seen in sixteen clumps. The highest and average latitudes are $71.4^{\circ}$ and $37.8^{\circ}$, respectively. Fifty-one velocity components were obtained and then each was identified as a single clump. Thirty-three clumps were further mapped at 1$^\prime$ resolution and 54 dense cores were extracted. Among dense cores, the average excitation temperature $T_{\mathrm{ex}}$ of $^{12}$CO is 10.3 K. The average line widths of thermal and non-thermal velocity dispersions are $0.19$ km s$^{-1}$ and $0.46$ km s$^{-1}$ respectively, suggesting that these cores are dominated by turbulence. Distances of the HGal clumps given by Gaia dust reddening are about $120-360$ pc. The ratio of $X_{13}$/$X_{18}$ is significantly higher than that in the solar neighbourhood, implying that HGal gas has a different star formation history compared to the gas in the Galactic disk. HGal cores with sizes from $0.01-0.1$ pc show no notable Larson's relation and the turbulence remains supersonic down to a scale of slightly below $0.1$ pc. None of the HGal cores which bear masses from 0.01-1 $M_{\odot}$ are gravitationally bound and all appear to be confined by outer pressure., 35 pages, 13 figures

During the transition phase from a prestellar to a protostellar cloud core, one or several protostars can form within a single gas core. The detailed physical processes of this transition, however, remain unclear. We present 1.3 mm dust continuum and molecular line observations with the Atacama Large Millimeter/submillimeter Array toward 43 protostellar cores in the Orion molecular cloud complex (λ Orionis, Orion B, and Orion A) with an angular resolution of ∼0.″35 (∼140 au). In total, we detect 13 binary/multiple systems. We derive an overall multiplicity frequency (MF) of 28% ± 4% and a companion star fraction (CSF) of 51% ± 6%, over a separation range of 300-8900 au. The median separation of companions is about 2100 au. The occurrence of stellar multiplicity may depend on the physical characteristics of the dense cores. Notably, those containing binary/multiple systems tend to show a higher gas density and Mach number than cores forming single stars. The integral-shaped filament of the Orion A giant molecular cloud (GMC), which has the highest gas density and hosts high-mass star formation in its central region (the Orion Nebula cluster), shows the highest MF and CSF among the Orion GMCs. In contrast, the λ Orionis GMC has a lower MF and CSF than the Orion B and Orion A GMCs, indicating that feedback from H ii regions may suppress the formation of multiple systems. We also find that the protostars comprising a binary/multiple system are usually at different evolutionary stages., T.L. acknowledges support from the National Natural Science Foundation of China (NSFC) through grants No. 12073061 and No. 12122307, the International Partnership Program of the Chinese Academy of Sciences (CAS) through grant No. 114231KYSB20200009, the Shanghai Pujiang Program (20PJ1415500), and science research grants from the China Manned Space Project with no. CMS-CSST-2021-B06. K.T. was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (grant No. 20H05645). D.J. and J.d.F. are supported by NRC Canada and by NSERC Discovery Grants. C.-F.L. acknowledge grants from the Ministry of Science and Technology of Taiwan (MoST 107-2119-M-001-040-MY3 and 110-2112-M-001-021-MY3) and Academia Sinica (Investigator Award AS-IA-108-M01). This research was carried out in part at the Jet Propulsion Laboratory, which is operated by the California Institute of Technology under a contract with the National Aeronautics and Space Administration (80NM0018D0004). J.-E.L. was supported by a National Research Foundation of Korea grant funded by the Korean government (MSIT) (grant No. 2021R1A2C1011718). J.H. acknowledges the support of NSFC projects 11873086 and U1631237. This work is sponsored (in part) by the CAS, through a grant to the CAS South America Center for Astronomy in Santiago, Chile. S.-L.Q. is supported by the NSFC with grant No. 12033005. S.Z. acknowledges the support of the China Postdoctoral Science Foundation through grant No. 2021M700248. L.B. gratefully acknowledges support by the ANID BASAL projects ACE210002 and FB210003. P.S. was supported by a Grant-in-Aid for Scientific Research (KAKENHI No. 18H01259) of JSPS. V.-M.P. acknowledges support by the grant PID2020-115892GB-I00 funded by MCIN/AEI/10.13039/501100011033.

We have selected six sources (G209.55–19.68S2, G205.46–14.56S1-A, G203.21–11.20W2, G191.90–11.21S, G205.46–14.56S3, and G206.93–16.61W2) from the Atacama Large Millimeter/submillimeter Array Survey of Orion Planck Galactic Cold Clumps (ALMASOP), in which these sources have been mapped in the CO (J = 2−1), SiO (J = 5−4), and C18O (J = 2−1) lines. These sources have high-velocity SiO jets surrounded by low-velocity CO outflows. The SiO jets consist of a chain of knots. These knots have been thought to be produced by semiperiodic variations in jet velocity. Therefore, we adopt a shock-forming model, which uses such variations to estimate the inclination angle and velocity of the jets. We also derive the inclination angle of the CO outflows using the wide-angle wind-driven shell model and find it to be broadly consistent with that of the associated SiO jets. In addition, we apply this shock-forming model to another three protostellar sources with SiO jets in the literature—HH 211, HH 212, and L1448C(N)—and find that their inclination angle and jet velocity are consistent with those previously estimated from proper-motion and radial-velocity studies.

Spike (S) glycoprotein of SARS-CoV2 exists chiefly in two conformations, open and closed. Most previous structural studies on S protein have been conducted at pH 8.0, but knowledge of the conformational propensities under both physiological and endosomal pH conditions is important to inform vaccine development. Our current study employed single-particle cryoelectron microscopy to visualize multiple states of open and closed conformations of S protein at physiological pH 7.4 and near-physiological pH 6.5 and pH 8.0. Propensities of open and closed conformations were found to differ with pH changes, whereby around 68% of S protein exists in open conformation at pH 7.4. Furthermore, we noticed a continuous movement in the N-terminal domain, receptor-binding domain (RBD), S2 domain, and stalk domain of S protein conformations at various pH values. Several key residues involving RBD-neutralizing epitopes are differentially exposed in each conformation. This study will assist in developing novel therapeutic measures against SARS-CoV2., Graphical abstract, In this study, Pramanick et al. demonstrate inherent structural flexibility of NTD, RBD, and stalk domain of SARS-CoV2 Spike glycoprotein. Eleven high-resolution cryo-EM structures obtained over a range of near-physiological pH values indicate a trend of increasing open conformational state of S protein at physiological pH 7.4.

We present here identification and characterization of the young stellar population associated with an active star-forming site Sh2-242. We used our own new optical imaging and spectroscopic observational data, as well as several archival catalogs, e.g., Pan-STARRS 1, $Gaia$ DR2, IPHAS, WIRCam, 2MASS, and $Spitzer$. Slit spectroscopic results confirm the classification of the main ionizing source BD+26 980 as an early-type star of spectral type B0.5 V. The spectrophotometric distance of the star is estimated as 2.08 $\pm$ 0.24 kpc, which confirms the source as a member of the cluster. An extinction map covering a large area (diameter $\sim$ 50') is generated with $H$ and $K$ photometry toward the region. From the map, three distinct locations of peak extinction complexes ($A_{V}$ $\simeq$ 7$-$17 mag) are identified for the very first time. Using the infrared color excess, a total of 33 Class I and 137 Class II young objects are classified within the region. The IPHAS photometry reveals classification of 36 H$\alpha$ emitting sources, which might be class II objects. Among 36 H$\alpha$ emitting sources, 5 are already identified using infrared excess emission. In total, 201 young objects are classified toward S242 from this study. The membership status of the young sources is further windowed with the inclusion of parallax from the $Gaia$ DR2 catalog. Using the optical and infrared color-magnitude diagrams, the young stellar objects are characterized with an average age of $\sim$ 1 Myr and the masses in the range 0.1$-$3.0 $M_\odot$. The census of the stellar content within the region is discussed using combined photometric and spectroscopic data., Comment: 17 pages, 12 figures, Published in the Astronomical Journal

The presence of complex organic molecules (COMs) in the interstellar medium (ISM) is of great interest since it may link to the origin and prevalence of life in the universe. Aiming to investigate the occurrence of COMs and their possible origins, we conducted a chemical census toward a sample of protostellar cores as part of the ALMA Survey of Orion Planck Galactic Cold Clumps (ALMASOP) project. We report the detection of 11 hot corino sources, which exhibit compact emissions from warm and abundant COMs, among 56 Class 0/I protostellar cores. All the hot corino sources discovered are likely Class 0 and their sizes of the warm region ($>$ 100 K) are comparable to 100 au. The luminosity of the hot corino sources exhibits positive correlations with the total number of methanol and the extent of its emissions. Such correlations are consistent with the thermal desorption picture for the presence of hot corino and suggest that the lower luminosity (Class 0) sources likely have a smaller region with COMs emissions. With the same sample selection method and detection criteria being applied, the detection rates of the warm methanol in the Orion cloud (15/37) and the Perseus cloud (28/50) are statistically similar when the cloud distances and the limited sample size are considered. Observing the same set of COM transitions will bring a more informative comparison between the cloud properties., 34 pages. 14 figures. 1 figure set. 1 machine-readable table. Accepted for publication in ApJ

Jets and outflows trace the accretion history of protostars. High-velocity molecular jets have been observed from several protostars in the early Class 0 phase of star formation, detected with the high-density tracer SiO. Until now, no clear jet has been detected with SiO emission from isolated evolved Class I protostellar systems. We report a prominent dense SiO jet from a Class I source G205S3 (HOPS-315: T bol ∼ 180 K, spectral index ∼0.417), with a moderately high mass-loss rate (∼0.59 × 10−6 M ⊙ yr−1) estimated from CO emission. Together, these features suggest that G205S3 is still in a high-accretion phase, similar to that expected of Class 0 objects. We compare G205S3 to a representative Class 0 system G206W2 (HOPS-399) and literature Class 0/I sources to explore the possible explanations behind the SiO emission seen at the later phase. We estimate a high inclination angle (∼40°) for G205S3 from CO emission, which may expose the infrared emission from the central core and mislead the spectral classification. However, the compact 1.3 mm continuum, C18O emission, location in the bolometric luminosity to submillimeter fluxes diagram, outflow force (∼3.26 × 10−5 M ⊙ km s−1 yr−1) are also analogous to that of Class I systems. We thus consider G205S3 to be at the very early phase of Class I, and in the late phase of high accretion. The episodic ejection could be due to the presence of an unknown binary, a planetary companion, or dense clumps, where the required mass for such high accretion could be supplied by a massive circumbinary disk.

We report here CCD $I$-band time-series photometry of a young (2$-$5 Myr) cluster NGC 2282 to identify and understand the variability of pre-main-sequence (PMS) stars. The $I$-band photometry, down to $\sim$ 20.5 mag, enables us to probe the variability towards the lower mass end ($\sim$ 0.1 M$_\odot$) of the PMS stars. From the light curves of 1627 stars, we identified 62 new photometric variable candidates. Their association with the region was established from H$\alpha$ emission and infrared (IR) excess. Among 62 variables, 30 young variables exhibit H$\alpha$ emission, near-IR (NIR)/mid-IR (MIR) excess or both, and they are candidate members of the cluster. Out of 62 variables, 41 are periodic variables with the rotation rate ranging from 0.2 to 7 days. The period distribution exhibits a median period at $\sim$ 1-day as in many young clusters (e.g., NGC~2264, ONC, etc.), but it follows a uni-modal distribution unlike others having bimodality with the slow rotators peaking at $\sim$ 6$-$8 days. To investigate the rotation-disk and variability-disk connection, we derived NIR excess from $\Delta$(I$-$K) and MIR excess from $Spitzer$ [3.6]$-$[4.5] $\mu$m data. No conclusive evidence of slow rotation with the presence of disks around stars and fast rotation for diskless stars is obtained from our periodic variables. A clear increasing trend of the variability amplitude with the IR excess is found for all variables.

The ESCRT pathway functions at different subcellular membranes to induce their negative curvature, and it has been largely characterized in model eukaryotes belonging to Opisthokonta. But searches of the genomes of many nonopisthokonts belonging to various supergroups indicate that some of them may harbour fewer ESCRT components. Of the genomes explored thus far, one of the most minimal set of ESCRT components was identified in the human pathogen Giardia lamblia, which belongs to Excavata. Here we report that an ESCRT-mediated pathway most likely operates at the peripheral vesicles, which are located at the cell periphery and the bare zone of this protist. Functional comparison of all the identified putative giardial ESCRT components, with the corresponding well-characterized orthologues from Saccharomyces cerevisiae, indicated that only some of the ESCRT components could functionally substitute for the corresponding yeast proteins. While GlVps25, GlVps2, and all three paralogues of GlVps4, tested positive in functional complementation assays, GlVps22, GlVps20, and GlVps24 did not. Binary interactions of either GlVps22 or GlVps25, with other ESCRT-II components from Giardia or yeast indicate that the giardial Vps36 orthologue is either completely missing or highly diverged. Interactions within the giardial ESCRT-III components also differ from those in yeast; while GlVps46a interacts preferentially with Vps24 compared to Vps2, GlVps46b, like the yeast orthologue, interacts with both.

Thermostable direct hemolysin (TDH) of

We present optical $I$-band light curves of the stars towards a star-forming region Cygnus OB7 from 17 nights photometric observations. The light curves are generated from a total of 381 image frames with very good photometric precision. From the light curves of 1900 stars and their periodogram analyses, we detect 31 candidate variables including five previously identified. 14 out of 31 objects are periodic and exhibit the rotation rates in the range of 0.15 to 11.60 days. We characterize those candidate variables using optical/infrared colour-colour and colour-magnitude diagrams. From spectral indices of the candidate variables, it turns out that four are probably Classical T-Tauri stars (CTTSs), rest remain unclassified from present data, they are possibly field stars or diskless pre-main sequence stars towards the region. Based on their location on the various colour-magnitude diagrams, the ages of two T Tauri Stars were estimated to be $\sim$ 5 Myr. The light curves indicate at least five of the periodic variables are eclipsing systems. The spatial distribution of young variable candidates on Planck 857 GHz (350 $\mu$m) and 2MASS $K_s$ images suggest that at least two of the CTTSs are part of the active star-forming cloud Lynds 1003., Comment: 14 pages, 12 figures

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

We report the detection of four new hot corino sources, G211.47-19.27S, G208.68-19.20N1, G210.49-19.79W and G192.12-11.10 from a survey study of Planck Galactic Cold Clumps in the Orion Molecular Cloud Complex with the Atacama Compact Array (ACA). Three sources had been identified as low mass Class 0 protostars in the Herschel Orion Protostar Survey (HOPS). One source in the lambda Orionis region is firstly reported as a protostellar core. We have observed abundant complex organic molecules (COMs), primarily methanol but also other oxygen-bearing COMs (in G211.47-19.27S and G208.68-19.20N1) and the molecule of prebiotic interest NH2CHO (in G211.47-19.27S), signifying the presence of hot corinos. While our spatial resolution is not sufficient for resolving most of the molecular emission structure, the large linewidth and high rotational temperature of COMs suggest that they likely reside in the hotter and innermost region immediately surrounding the protostar. In G211.47-19.27S, the D/H ratio of methanol ([CH2DOH]/[CH3OH]) and the 12C/13C ratio of methanol ([CH3OH]/[13CH3OH]) are comparable to those of other hot corinos. Hydrocarbons and long carbon-chain molecules such as c-C3H2 and HCCCN are also detected in the four sources, likely tracing the outer and cooler molecular envelopes., 37 pages, 51 figures, to be published in ApJ

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite neutralizers are potential candidates for isolation of HIV-1 bNAbs. The coexistence of bNAbs such as BG18 with neutralization-susceptible autologous viruses in an HIV-1-infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1-infected children than in adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral-naive HIV-1 clade C chronically infected pediatric elite neutralizer, AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, a median inhibitory dilution (ID(50)) value of 1,246, and presence of N160 and N332 supersite-dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses, exhibited substantial indels despite limited somatic hypermutations, interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy, and demonstrated high binding affinity. In addition, AIIMS-P01 neutralized the coexisting and evolving autologous viruses, suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in the AIIMS_330 pediatric elite neutralizer. Such pediatric elite neutralizers can serve as potential candidates for isolation of novel HIV-1 pediatric bNAbs and for understanding the coevolution of virus and host immune response. IMPORTANCE More than 50% of the HIV-1 infections globally are caused by clade C viruses. To date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are under way to design immunogens that can elicit correlates of protection upon vaccination. Here, we report the isolation and characterization of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01, from a clade C chronically infected pediatric elite neutralizer. The N332 supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibited substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation and characterization of bNAbs contributing to the plasma breadth in HIV-1 chronically infected children may help provide a better understanding of their role in controlling HIV-1 infection.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones present ubiquitously in all kingdoms of life. Their low molecular weight subunits associate to form higher order structures. Under conditions of stress, sHSPs prevent aggregation of substrate proteins by undergoing rapid changes in their conformation or stoichiometry. Polydispersity and dynamic nature of these proteins have made structural investigations through crystallography a daunting task. In pathogens like Mycobacteria, sHSPs are immuno-dominant antigens, enabling survival of the pathogen within the host and contributing to disease persistence. We characterized sHSPs from Mycobacterium marinum M and determined the crystal structure of one of these. The protein crystallized in three different conditions as dodecamers, with dimers arranged in a tetrahedral fashion to form a closed cage-like architecture. Interestingly, we found a pentapeptide bound to the dodecamers revealing one of the modes of sHSP-substrate interaction. Further, we have observed that ATP inhibits the chaperoning activity of the protein.

The wave of resolution revolution in cryo-EM has touched, and made a significant impact on, the structural biology of GPCRs. High-resolution structures of several GPCR-G-protein complexes are now determined by cryo-EM and they illuminate fine structural details of this central macromolecular complex involved in cellular signaling.

Binding to the von Willebrand factor (VWF) D′D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D′D3 alone and in complex with FVIII. The D′D3 dimer ([D′D3]2) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D′. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D′ domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. Modeling the FVIII structure into the three-dimensional EM reconstructions of [D′D3]2-FVIII ternary and quaternary complexes indicates conformational rearrangements of the FVIII C domains compared with their disposition in the unbound state. These results illustrate the cooperative plasticity between VWF and FVIII that coordinate their high-affinity interaction.

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems. Polyketide synthases are multidomain enzymes that produce polyketides, which form the basis of many therapeutic agents; here, electron cryo-microscopy is used to establish the structure of a bacterial full-length module, and to elucidate the structural basis of both intramodule and intermodule substrate transfer. Polyketide synthases (PKSs) are multi-domain enzyme complexes producing polyketides, a large class of secondary metabolites — in other words, natural products. Two papers from Georgios Skiniotis and colleagues use cryo-electron microscopy to probe the structure of an intact module of a full-length multienzyme PKS module involved in pikromycin biosynthesis in the bacterium Streptomyces venezuelae in different functional states. The structures reveal how the ketosynthase, acyltransferase, ketoreductase and acyl carrier protein (ACP) domains interact during the catalytic cycle. In each state the ACP is differentially positioned to facilitate intermediate transfer for the next catalytic step and for transfer to the next module.

Polyketide synthases (PKSs) are multidomain enzymes that produce polyketides, which form the basis of many therapeutic agents; here, electron cryo-microscopy is used to probe the structure of an intact module of a multi-enzyme PKS in different functional states. Polyketide synthases (PKSs) are multi-domain enzyme complexes producing polyketides, a large class of secondary metabolites — in other words, natural products. Two papers from Georgios Skiniotis and colleagues use cryo-electron microscopy to probe the structure of an intact module of a full-length multienzyme PKS module involved in pikromycin biosynthesis in the bacterium Streptomyces venezuelae in different functional states. The structures reveal how the ketosynthase, acyltransferase, ketoreductase and acyl carrier protein (ACP) domains interact during the catalytic cycle. In each state the ACP is differentially positioned to facilitate intermediate transfer for the next catalytic step and for transfer to the next module. The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents1. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper2). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.

The surname of author Toon Laeremans was misspelled ‘Laermans’. This error has been corrected online.