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Structural genomics began as a global effort in the 1990s to determine the tertiary structures of all protein families as a response to large-scale genome sequencing projects. The immediate outcome was an influx of tens of thousands of protein structures, many of which had unknown functions. At the time, the value of structural genomics was controversial. However, the structures themselves were only the most obvious output. In addition, these newly solved structures motivated the emergence of huge data science and infrastructure efforts, which, together with advances in Deep Learning, have brought about a revolution in computational molecular biology. Here, we review some of the computational research carried out at the Protein Data Bank Japan (PDBj) during the Protein 3000 project under the leadership of Haruki Nakamura, much of which continues to flourish today.

Chordotonal neurons are responsible for sound sensation in Drosophila. However, little is known about how they respond to sound with high sensitivity. Using genetic labeling, we found one of the Drosophila axonemal dynein heavy chains, CG9492 (DNAH5), was specifically expressed in larval chordotonal neurons and showed a distribution restricted to proximal cilia. While DNAH5 mutation did not affect the cilium morphology or the trafficking of Inactive, a candidate auditory transduction channel, larvae with DNAH5 mutation had reduced startle responses to sound at low and medium intensities. Calcium imaging confirmed that DNAH5 functioned autonomously in chordotonal neurons for larval sound sensation. Furthermore, disrupting DNAH5 resulted in a decrease of spike firing responses to low-level sound in chordotonal neurons. Intriguingly, DNAH5 mutant larvae displayed an altered frequency tuning curve of the auditory organs. All together, our findings support a critical role of DNAH5 in tuning the frequency selectivity and the sound sensitivity of larval auditory neurons. SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-021-00631-w) contains supplementary material, which is available to authorized users.

Motivation The scoring of antibody–antigen docked poses starting from unbound homology models has not been systematically optimized for a large and diverse set of input sequences. Results To address this need, we have developed AbAdapt, a webserver that accepts antibody and antigen sequences, models their 3D structures, predicts epitope and paratope, and then docks the modeled structures using two established docking engines (Piper and Hex). Each of the key steps has been optimized by developing and training new machine-learning models. The sequences from a diverse set of 622 antibody–antigen pairs with known structure were used as inputs for leave-one-out cross-validation. The final set of cluster representatives included at least one ‘Adequate’ pose for 550/622 (88.4%) of the queries. The median (interquartile range) ranks of these ‘Adequate’ poses were 22 (5–77). Similar results were obtained on a holdout set of 100 unrelated antibody–antigen pairs. When epitopes were repredicted using docking-derived features for specific antibodies, the median ROC AUC increased from 0.679 to 0.720 in cross-validation and from 0.694 to 0.730 in the holdout set. Availability and implementation AbAdapt and related data are available at https://sysimm.org/abadapt/. Supplementary information Supplementary data are available at Bioinformatics Advances online.

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.One Sentence SummaryOmicron, carrying ∼30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.

Auditory transduction is mediated by chordotonal (Cho) neurons in Drosophila larvae, but the molecular identity of the mechanotransduction (MET) channel is elusive. Here, we established a whole-cell recording system of Cho neurons and showed that two transient receptor potential vanilloid (TRPV) channels, Nanchung (NAN) and Inactive (IAV), are essential for MET currents in Cho neurons. NAN and IAV form active ion channels when expressed simultaneously in S2 cells. Point mutations in the pore region of NAN-IAV change the reversal potential of the MET currents. Particularly, residues 857 through 990 in the IAV carboxyl terminus regulate the kinetics of MET currents in Cho neurons. In addition, TRPN channel NompC contributes to the adaptation of auditory transduction currents independent of its ion-conduction function. These results indicate that NAN-IAV, rather than NompC, functions as essential pore-forming subunits of the native auditory transduction channel in Drosophila and provide insights into the gating mechanism of MET currents in Cho neurons.

Breakthrough infection is often observed for the SARS-CoV-2 Delta variant, and neutralizing antibody levels are associated with vaccine efficiency1. Recent studies revealed that not only anti-receptor binding domain (RBD) antibodies2 but also antibodies against the N-terminal domain (NTD) play important roles in positively3,4 or negatively4-8 controlling SARS-CoV-2 infectivity. Here, we found that the Delta variant completely escaped from anti-NTD neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Cryo-EM analysis of the Delta spike revealed that epitopes for anti-NTD neutralizing antibodies are structurally divergent, whereas epitopes for enhancing antibodies are well conserved with wild-type spike protein. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the original Delta variant, when major anti-RBD neutralizing antibody epitopes remaining in the Delta variant were disrupted, some BNT162b2-immune sera not only lost neutralizing activity but became infection-enhanced. The enhanced infectivity disappeared when the Delta NTD was substituted with the wild-type NTD. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta variant lacking anti-RBD antibody epitopes without enhancing infectivity. Importantly, SARS-CoV-2 variants with similar mutations in the RBD have already emerged according to the GISAID database and their pseudoviruses were resistant to some BNT162b2-immune sera. These findings demonstrate that mutations in the NTD, as well as the RBD, play an important role in antibody escape by SARS-CoV-2. Development of effective vaccines against emerging variants will be necessary, not only to protect against infection, but also to prevent further mutation of SARS-CoV-2.

The COVID-19 (coronavirus disease 2019) pandemic is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). SARS-CoV-2 produces a small hydrophobic envelope (E) protein which shares high homology with SARS-CoV E protein. By patch-clamp recording, the E protein is demonstrated to be a cation-selective ion channel. Furthermore, the SARS-CoV-2 E protein can be blocked by a SARS-CoV E protein inhibitor hexamethylene amiloride. Using structural model and virtual screening, another E protein inhibitor AZD5153 is discovered. AZD5153 is a bromodomain protein 4 inhibitor against hematologic malignancies in clinical trial. The E protein amino acids Phe23 and Val29 are key determinants for AZD5153 sensitivity. This study provides two promising lead compounds and a functional assay of SARS-CoV-2 E protein for the future drug candidate discovery.

mRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.

Birds, reptiles and insects have the ability to discriminate humidity levels that influence their survival and geographic distribution. Insects are particularly susceptible to humidity changes due to high surface area to volume ratios, but it remains unclear how humidity sensors transduce humidity signals. Here we identified Or42b-expressing olfactory sensory neurons, which are required for moisture attraction in Drosophila. The sensilla housing Or42b neurons show cuticular deformations upon moist air stimuli, indicating a conversion of humidity into mechanical force. Accordingly, we found Or42b neurons directly respond to humidity changes and rely on the mechanosensitive ion channel TMEM63 to mediate humidity sensing (hygrosensation). Expressing human TMEM63B in Tmem63 mutant flies rescued their defective phenotype in moisture attraction, demonstrating functional conservation. Thus, our results reveal a role of Tmem63 in hygrosensation and support the strategy to detect humidity by transforming it into a mechanical stimulus, which is unique in sensory transduction.

The repertoires of B cell receptors (BCRs), which can be captured by single cell-resolution sequencing technologies, contain a personal history of a donor's antigen exposure. One of the current challenges in analyzing such BCR sequence data is to assign sequences to groups with similar antigen and epitope binding specificity. This is a non-trivial task given the paucity of experimentally-determined antibody–antigen structures and the fact that different gene combinations in B cells can lead to receptors that target the same antigen and epitope. Here, we describe a method for clustering BCRs based on sequence and predicted structural features in order to predict groups with similar antigen and epitope binding specificity. We show that all known experimentally-determined structures of antibody–antigen complexes can be clustered accurately (AUC 0.981) and that use of predicted structural features improved the accuracy of the epitope classification. We next show that an independent and non-redundant set of 104 anti-HIV antibody sequences could be clustered corresponding to manually-assigned epitopes with a specificity of 99.7% and a sensitivity of 61.93%, with the imbalance in sensitivity due almost entirely to one group of antibodies—those that target the gp120 V3 loop, which do not form a single, well-defined cluster. We next examined a diverse set of anti-hemagglutinin BCR sequences from humans and mice. We observed clusters that included human or mouse sequences with anti-hemagglutinin antibodies of known structure. We also observed clusters that included both human and mouse sequences. Importantly, to the extent that the epitopes have been experimentally characterized, none of the observed clusters erroneously grouped different hemagglutinin binding regions. Taken together, these results demonstrate that the proposed clustering method provides high-throughput prediction of BCRs with common binding specificity across clonal lineages, donors and even species.

Repertoire Builder (https://sysimm.org/rep_builder/) is a method for generating atomic-resolution, three-dimensional models of B cell receptors (BCRs) or T cell receptors (TCRs) from their amino acid sequences. It is currently capable of handling batches of up to 104 sequences in approximately 30 minutes. This performance was achieved by applying a multiple sequence alignment extension technique originally developed for phylogenetic analysis to the template selection problem of complementarity determining region (CDR) loops. Under comparable conditions, average all-atom root-mean square deviations (RMSDs) from experimentally-determined structures of CDRH3 loops in BCRs were significantly lower than tested third-party high-throughput modeling methods, including ABodyBuilder, PigsPro, and LYRA. For TCRs, similar trends were observed when Repertoire Builder was compared with TCRmodel and LYRA. We also found that Repertoire Builder model errors were, in general, lower than those produced by our earlier Kotai Antibody Builder, even when CDRH3 loop refinement was used. However, in a subset of cases, which could be distinguished by poor Repertoire Builder scores, refinement by Kotai Antibody Builder or Rosetta Antibody, both of which utilize extensive structural sampling, improved the third heavy chain CDR (CDRH3) RMSD on average. Taken together, these results indicate that the MSA extension approach used by Repertoire Builder resulted in a favorable balance between speed and accuracy when compared to alternative methods. Furthermore, we conclude that more sensitive scoring, rather than extended structural sampling, is needed to further improve the accuracy of BCR and TCR modeling.

Graphical abstract, B cell receptors (BCRs) and T cell receptors (TCRs) make up an essential network of defense molecules that, collectively, can distinguish self from non-self and facilitate destruction of antigen-bearing cells such as pathogens or tumors. The analysis of BCR and TCR repertoires plays an important role in both basic immunology as well as in biotechnology. Because the repertoires are highly diverse, specialized software methods are needed to extract meaningful information from BCR and TCR sequence data. Here, we review recent developments in bioinformatics tools for analysis of BCR and TCR repertoires, with an emphasis on those that incorporate structural features. After describing the recent sequencing technologies for immune receptor repertoires, we survey structural modeling methods for BCR and TCRs, along with methods for clustering such models. We review downstream analyses, including BCR and TCR epitope prediction, antibody-antigen docking and TCR-peptide-MHC Modeling. We also briefly discuss molecular dynamics in this context.

The leakage failure of the austenitic stainless steel tubes in the hydrogenation unit of a petrochemical company was analyzed. The causes of the failure were investigated by visual examination, chemical compositions analysis of the tube, microscopic examination, metallographic examination and scale layer analysis. The results showed that the directly reason of the failure is pitting corrosion and stress corrosion cracking. We concluded that the improper heat treatment of the austenitic stainless tubes and existence of the corrosive substances including wet hydrogen sulfide and hydrogen chloride in the working medium, result in the occurrence of the corrosion, and the complex flowing conditions accelerate the corrosion process. Proposals were put forward in this paper.

The AT-rich interactive domain-containing protein 5a (Arid5a) plays a critical role in autoimmunity by regulating the half-life of Interleukin-6 (IL-6) mRNA. However, the signaling pathways underlying Arid5a-mediated regulation of IL-6 mRNA stability are largely uncharacterized. Here, we found that during the early phase of lipopolysaccharide (LPS) stimulation, NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA. Subsequently, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) promotes translocation of AU-rich element RNA-binding protein 1 (AUF-1) from the nucleus to the cytoplasm, where it destabilizes Arid5a mRNA by binding to AU-rich elements in the 3΄ UTR. This results in downregulation of IL-6 mRNA expression. During the late phase of LPS stimulation, p38 MAPK phosphorylates Arid5a and recruits the WW domain containing E3 ubiquitin protein ligase 1 (WWP1) to its complex, which in turn ubiquitinates Arid5a in a K48-linked manner, leading to its degradation. Inhibition of Arid5a phosphorylation and degradation increases production of IL-6 mRNA. Thus, our data demonstrate that LPS-induced NF-κB and MAPK signaling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. This study therefore substantially increases our understanding of the mechanisms by which IL-6 is regulated.

One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5’UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5’UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure.Author summaryOne of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA, which is required for efficient propagation. Recently, it has been reported that interaction of miR-122 with the 5’UTR of HCV contributes to the folding of a functional IRES structure that is required for efficient translation of viral RNA. In this study, we examined the minimum motifs in the seed region of miRNAs required for the enhancement of HCV replication. As a result, we found two groups of non-hepatic miRNAs: “miR-122-like miRNAs” that can bind HCV-RNA at two locations in a manner similar to miR-122, and “non-miR-122-like miRNAs” that target a single site that masking both G28 and C29 in the 5’UTR. The interaction of these non-hepatic miRNAs with the 5’UTR can facilitate not only the folding of active HCV IRES but also the stabilization of IRES 3D structure in order to facilitate binding to the ribosome. These results suggest the possibility of replication of HCV in non-hepatic cells through interaction with miRNAs other than miR-122 and provide insight into the establishment of persistent infection of HCV in non-hepatic tissues that lead to the development of extrahepatic manifestations.

Here, we describe a web server that integrates structural alignments with the MAFFT multiple sequence alignment (MSA) tool. For this purpose, we have prepared a web-based Database of Aligned Structural Homologs (DASH), which provides structural alignments at the domain and chain levels for all proteins in the Protein Data Bank (PDB), and can be queried interactively or by a simple REST-like API. MAFFT-DASH integration can be invoked with a single flag on either the web (https://mafft.cbrc.jp/alignment/server/) or command-line versions of MAFFT. In our benchmarks using 878 cases from the BAliBase, HomFam, OXFam, Mattbench and SISYPHUS datasets, MAFFT-DASH showed 10–20% improvement over standard MAFFT for MSA problems with weak similarity, in terms of Sum-of-Pairs (SP), a measure of how well a program succeeds at aligning input sequences in comparison to a reference alignment. When MAFFT alignments were supplemented with homologous sequences, further improvement was observed. Potential applications of DASH beyond MSA enrichment include functional annotation through detection of remote homology and assembly of template libraries for homology modeling.

Antibodies against the receptor-binding-domain of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal-domain (NTD) induced the open conformation of receptor binding domain (RBD) and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all the infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection., A subset antibodies that are detected in patients with severe COVID-19 target a specific region of the N terminal domain of the spike protein and enhance binding of the virus to the ACE2 receptor.

One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5’UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5’UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure., Author summary One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA, which is required for efficient propagation. Recently, it has been reported that interaction of miR-122 with the 5’UTR of HCV contributes to the folding of a functional IRES structure that is required for efficient translation of viral RNA. In this study, we examined the minimum motifs in the seed region of miRNAs required for the enhancement of HCV replication. As a result, we found two groups of non-hepatic miRNAs: “miR-122-like miRNAs” that can bind HCV-RNA at two locations in a manner similar to miR-122, and “non-miR-122-like miRNAs” that target a single site that masking both G28 and C29 in the 5’UTR. The interaction of these non-hepatic miRNAs with the 5’UTR can facilitate not only the folding of active HCV IRES but also the stabilization of IRES 3D structure in order to facilitate binding to the ribosome. These results suggest the possibility of replication of HCV in non-hepatic cells through interaction with miRNAs other than miR-122 and provide insight into the establishment of persistent infection of HCV in non-hepatic tissues that lead to the development of extrahepatic manifestations.

The evolutional process of disease-associated autoantibodies in systemic lupus erythematosus (SLE) remains to be established. Here we show intraclonal diversification and affinity maturation of anti-nuclear antibody (ANA)-producing B cells in SLE. We identified a panel of monoclonal ANAs recognizing nuclear antigens, such as double-stranded DNA (dsDNA) and ribonucleoproteins (RNPs) from acute SLE subjects. These ANAs had relatively few, but nonetheless critical mutations. High-throughput immunoglobulin sequencing of blood lymphocytes disclosed the existence of sizable ANA lineages shearing critical mutations intraclonally. We further focused on anti-DNA antibodies, which are capable to bind to both single-stranded (ss) and dsDNA at high affinity. Crystal structure and biochemical analysis confirmed a direct role of the mutations in the acquisition of DNA reactivity and also revealed that these anti-DNA antibodies recognized an unpaired region within DNA duplex. Our study unveils the unique properties of high-affinity anti-DNA antibodies that are generated through antigen-driven affinity maturation in acute phase of SLE.

The mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in regulating cell growth and metabolism by responding to cellular nutrient conditions. The activity of mTORC1 is controlled by Rag GTPases, which are anchored to lysosomes via Ragulator, a pentameric protein complex consisting of membrane-anchored p18/LAMTOR1 and two roadblock heterodimers. Here we report the crystal structure of Ragulator in complex with the roadblock domains of RagA-C, which helps to elucidate the molecular basis for the regulation of Rag GTPases. In the structure, p18 wraps around the three pairs of roadblock heterodimers to tandemly assemble them onto lysosomes. Cellular and in vitro analyses further demonstrate that p18 is required for Ragulator-Rag GTPase assembly and amino acid-dependent activation of mTORC1. These results establish p18 as a critical organizing scaffold for the Ragulator-Rag GTPase complex, which may provide a platform for nutrient sensing on lysosomes., mTORC1 activity is controlled through Rag GTPases, which are anchored to the lysosome through the Ragulator. Here, the authors give molecular insights into Ragulator-Rag GTPase assembly and present the crystal structures of the Ragulator alone and in complex with the RagA-C roadblock domains.

To achieve a high yield of tropane alkaloids (TA) and exploit the alpine plant sustainably, an optimized protocol for induction and establishment of hairy roots culture of Prezwalskia tangutica Maxim was developed through selection of appropriate Agrobacterium strain and the explant type. The hypocotyl is more readily facile to induce the HR than the cotyledon is when infected with the three different agrobacterium strains. MUS440 has an efficiency (of up to 20%), whereas the ATCC10060 (A4) can induce HR on both types of explants with the highest frequency (33.33%), root length (21.17 ± 2.84 cm), and root number (10.83 ± 1.43) per explant than the other strains. The highest HR production resulted from using hypocotyl as explants. Independent transformed HR was able to grow vigorously and to propagate on a no-hormone 1/2MS liquid medium. The presence of pRi rolB gene in transformation of HR was confirmed by PCR amplification. In the liquid medium, the HR growth curve appeared to be “S” shaped, and ADB had increased to 4.633 g/l. Moreover, HPLC analysis showed that HR lines have an extraordinary ability to produce atropine (229.88 mg/100 g), anisodine (4.09 mg/100 g), anisodamine (12.85 mg/100 g), and scopolamine (10.69 mg/100 g), which were all more significant than the control roots. In conclusion, our study optimized the culture condition and established a feasible genetics reactor for P. tangutica green exploration and biological study in the alpine region.

Increasing awareness of the importance of protein–RNA interactions has motivated many approaches to predict residue-level RNA binding sites in proteins based on sequence or structural characteristics. Sequence-based predictors are usually high in sensitivity but low in specificity; conversely structure-based predictors tend to have high specificity, but lower sensitivity. Here we quantified the contribution of both sequence- and structure-based features as indicators of RNA-binding propensity using a machine-learning approach. In order to capture structural information for proteins without a known structure, we used homology modeling to extract the relevant structural features. Several novel and modified features enhanced the accuracy of residue-level RNA-binding propensity beyond what has been reported previously, including by meta-prediction servers. These features include: hidden Markov model-based evolutionary conservation, surface deformations based on the Laplacian norm formalism, and relative solvent accessibility partitioned into backbone and side chain contributions. We constructed a web server called aaRNA that implements the proposed method and demonstrate its use in identifying putative RNA binding sites.

Transcription-induced chimaeric transcripts, the potential post-transcriptional processing products, might reflect the spatial proximity of actively transcribed genes co-localized in transcription factories. A growing number of expression data deposited in databases provide us with the raw material for screening such chimaeric transcripts and using them as the probes to identify interactions between genes in cis or in trans. Based on the high-quality chimaeric transcripts gleaned from human expression sequence tag data with selection criteria, we identified the patterns of inter- and intrachromosomal gene–gene interactions. On top the contact pattern from interchromosomal interactions, we also observed an exponential behaviour of the intrachromosomal interactions within a certain length scale, which is consistent with the independent experimental results from Hi-C screening and with the Random Loop Model. A compatible result is found for mouse. Transcription-induced chimaeric transcripts, most of which might be accidental products with trivial functions, shed light on the spatial organization of chromosomes. These inter- and intrachromosomal interactions might contribute to the compaction of chromosomes, their segregation and formation of the chromosome territories, and their spatial distribution within the nucleus.

What physical mechanism leads to organization of a highly condensed and confined circular chromosome? Computational modeling shows that confinement-induced organization is able to overcome the chromosome's propensity to mix by the formation of topological domains. The experimentally observed high precision of separate subcellular positioning of loci (located on different chromosomal domains) in Escherichia coli naturally emerges as a result of entropic demixing of such chromosomal loops. We propose one possible mechanism for organizing these domains: regulatory control defined by the underlying E. coli gene regulatory network requires the colocalization of transcription factor genes and target genes. Investigating this assumption, we find the DNA chain to self-organize into several topologically distinguishable domains where the interplay between the entropic repulsion of chromosomal loops and their compression due to the confining geometry induces an effective nucleoid filament-type of structure. Thus, we propose that the physical structure of the chromosome is a direct result of regulatory interactions. To reproduce the observed precise ordering of the chromosome, we estimate that the domain sizes are distributed between 10 and 700 kb, in agreement with the size of topological domains identified in the context of DNA supercoiling.