Assembly Domain Research Articles

Functional information integration in product development by using assembly features

Product development is a collaboration-intensive process resulting in a novel or enhanced product that satisfies the customers' needs. To meet those needs, functional requirements are defined, which ultimately determine the product's physical characteristics and manufacturing. However, the functional and other non-geometrical information becomes less noticeable as the process advances, since the primary representation of the product design in the latter stages of the product development is often a purely geometrical model, causing information fragmentation. This fragmentation hinders the collaboration between different stakeholders while neglecting valuable product information that could facilitate its manufacturing. Research to date has not yet provided a suitable way to link the geometrical model of a product with its non-geometrical information. Focused on the assembly domain, this work proposes a way to integrate functional information into the product's geometrical model by using assembly features, which could be employed in the latter stages to extract process constraints and additional product details relevant to assembly process planning, as shown in the developed case study. This paper provides new insights into the value of the assembly feature as a functional information carrier and a tool for improving the collaboration between different stakeholders during the product development process.

Abstract 2548: Responsive assembly of signaling domains at the plasma membrane

Abstract 2548: Responsive assembly of signaling domains at the plasma membrane

The Construction of Knowledge Graphs in the Aviation Assembly Domain Based on a Joint Knowledge Extraction Model

The aviation assembly domain, which is a complex system, involves the multi-dimensional information of parts, processes, tools, plants and operation projects. In order to assist the knowledge management from natural language text in the aircraft manufacturing process, this paper proposes the corresponding ontology scheme and the joint knowledge extraction model, which is necessary for construct the knowledge graph in the aviation assembly domain. The model is able to automated end-to-end construct knowledge graph. The proposed model, which is based on reinforcement learning approach and a novel labeling scheme, takes the constraint relationships between entities and relations as an important identification basis. The model does not rely on manual feature setting, while it greatly reduces the training cost. The proposed joint knowledge extraction model was testified from the practical scenarios of the general assembly and component assembly. The experimental results showed that the proposed model showed an excellent performance in the aviation assembly domain, with the F1-score of 89.71% for entities, the F1-score of 91.27% for relations, and the overall average F1-score of 82.41%. Based on the superior performance of the model, the knowledge graph of the general assembly and component assembly, which included 1, 308 pairs of triples composed of five kinds of entities and three kinds of relations, was further constructed in the aviation assembly domain.

Multistep molecular mechanism of amyloid-like aggregation of nucleic acid-binding domain of TDP-43.

TDP-43 protein is associated with many neurodegenerative diseases and has been shown to adopt various oligomeric and fibrillar states. However, a detailed kinetic understanding of the structural transformation of the native form of the protein to the fibrillar state is missing. In this study, we delineate the temporal sequence of structural events during the amyloid-like assembly of the functional nucleic acid-binding domain of TDP-43. We kinetically mapped the aggregation process using multiple probes such as tryptophan and thioflavin T (ThT) fluorescence, circular dichroism (CD), and dynamic light scattering (DLS) targeting different structural events. Our data reveal that aggregation occurs in four distinct steps-very fast, fast, slow, and very slow. The "very fast" change results in partially unfolded forms that undergo conformational conversion, oligomerization and bind to ThT in the "fast step" to form higher order intermediates (HOI). The temporal sequence of the formation of ThT binding sites and conformational conversion depends upon the protein concentration. The HOI further undergoes structural rearrangement to form protofibrils in the "slow" step, which, consequently, assembles in the "very slow" step to form an amyloid-like assembly. The spectroscopic properties of the amyloid-like assembly across the protein concentration remain similar. Additionally, we observe no lag phase across protein concentration for all the probes studied, suggesting that the aggregation process follows a linear polymerization reaction. Overall, our study demonstrates that the amyloid-like assembly forms in multiple steps, which is also supported by the temperature dependence of the kinetics.

Predicting the Assembly of the Transmembrane Domains of Viral Channel Forming Proteins and Peptide Drug Screening Using a Docking Approach.

A de novo assembly algorithm is provided to propose the assembly of bitopic transmembrane domains (TMDs) of membrane proteins. The algorithm is probed using, in particular, viral channel forming proteins (VCPs) such as M2 of influenza A virus, E protein of severe acute respiratory syndrome corona virus (SARS-CoV), 6K of Chikungunya virus (CHIKV), SH of human respiratory syncytial virus (hRSV), and Vpu of human immunodeficiency virus type 2 (HIV-2). The generation of the structures is based on screening a 7-dimensional space. Assembly of the TMDs can be achieved either by simultaneously docking the individual TMDs or via a sequential docking. Scoring based on estimated binding energies (EBEs) of the oligomeric structures is obtained by the tilt to decipher the handedness of the bundles. The bundles match especially well for all-atom models of M2 referring to an experimentally reported tetrameric bundle. Docking of helical poly-peptides to experimental structures of M2 and E protein identifies improving EBEs for positively charged (K,R,H) and aromatic amino acids (F,Y,W). Data are improved when using polypeptides for which the coordinates of the amino acids are adapted to the Cα coordinates of the respective experimentally derived structures of the TMDs of the target proteins.

Ion Transport in 2D Nanostructured π-Conjugated Thieno[3,2-b]thiophene-Based Liquid Crystal.

Leveraging the self-assembling behavior of liquid crystals designed for controlling ion transport is of both fundamental and technological significance. Here, we have designed and prepared a liquid crystal that contains 2,5-bis(thien-2-yl)thieno[3,2-b]thiophene (BTTT) as mesogenic core and conjugated segment and symmetric tetra(ethylene oxide) (EO4) as polar side chains for ion-conducting regions. Driven by the crystallization of the BTTT cores, BTTT/dEO4 exhibits well-ordered smectic phases below 71.5 °C as confirmed by differential scanning calorimetry, polarized optical microscopy, temperature-dependent wide-angle X-ray scattering, and grazing incidence wide-angle X-ray scattering (GIWAXS). We adopted a combination of experimental GIWAXS and molecular dynamics (MD) simulations to better understand the molecular packing of BTTT/dEO4 films, particularly when loaded with the ion-conducting salt lithium bis(trifluoromethanesulfonyl)imide (LiTFSI). Ionic conduction of BTTT/dEO4 is realized by the addition of LiTFSI, with the material able to maintain smectic phases up to r = [Li+]/[EO] = 0.1. The highest ionic conductivity of 8 × 10-3 S/cm was attained at an intermedium salt concentration of r = 0.05. It was also found that ion conduction in BTTT/dEO4 is enhanced by forming a smectic layered structure with irregular interfaces between the BTTT and EO4 layers and by the lateral film expansion upon salt addition. This can be explained by the enhancement of the misalignment and configurational entropy of the side chains, which increase their local mobility and that of the solvated ions. Our molecular design thus illustrates how, beyond the favorable energetic interactions that drive the assembly of ion solvating domains, modulation of entropic effects can also be favorably harnessed to improve ion conduction.

Facile synthesis of sulfonated poly(phenyl-alkane)s for proton exchange membrane fuel cells

Sulfonated poly (phenyl-alkane)s with all-carbon backbones exhibit excellent chemical stability; however, their applications in proton exchange membranes are limited. Herein, we report a facile synthesis of three sulfonated poly (phenyl-alkane)s SP1–SP3 via efficient acid-catalyzed Friedel−Crafts polycondensations of commercially available 2,3,4,5,6-pentafluorobenzaldehyde with three aromatic monomers, followed by mild and efficient nucleophilic substitution of para-aryl-F with sodium 4-oxybenzenesulfonate. In the characterization of the three polymers, the single-component adsorption–desorption isotherms of N2 and CO2 confirmed their intrinsic ultramicroporosity. For the SP3 membrane, small-angle X-ray scattering and atomic force microscopy measurements revealed that larger ionic clusters and smaller hydrophobic assembly domains led to better ionic channel connectivity. These features and the observed high ion exchange capacity and narrowly distributed ultramicropores synergistically determined its high conductivity. Moreover, the SP3 membrane exhibited excellent oxidative stability and good mechanical properties, enabling the successful application in H2/air proton exchange membrane fuel cells.

Double-strand break repair and mis-repair in 3D

DNA double-strand breaks (DSBs) are lesions that arise frequently from exposure to damaging agents as well as from ongoing physiological DNA transactions. Mis-repair of DSBs leads to rearrangements and structural variations in chromosomes, including insertions, deletions, and translocations implicated in disease. The DNA damage response (DDR) limits pathologic mutations and large-scale chromosome rearrangements. DSB repair initiates in 2D at DNA lesions with the stepwise recruitment of repair proteins and local chromatin remodeling which facilitates break accessibility. More complex structures are then formed via protein assembly into nanodomains and via genome folding into chromatin loops. Subsequently, 3D reorganization of DSBs is guided by clustering forces which drive the assembly of repair domains harboring multiple lesions. These domains are further stabilized and insulated into condensates via liquid-liquid phase-separation. Here, we discuss the benefits and risks associated with this 3D reorganization of the broken genome.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight β-strands and three α-helices with the topology α1-β1-β2-α2- β3- α3- β4- β5- β6- β7- β8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

Quaternary organization of the human eEF1B complex reveals unique multi-GEF domain assembly.

Protein synthesis in eukaryotic cell is spatially and structurally compartmentalized that ensures high efficiency of this process. One of the distinctive features of higher eukaryotes is the existence of stable multi-protein complexes of aminoacyl-tRNA synthetases and translation elongation factors. Here, we report a quaternary organization of the human guanine-nucleotide exchange factor (GEF) complex, eEF1B, comprising α, β and γ subunits that specifically associate into a heterotrimeric form eEF1B(αβγ)3. As both the eEF1Bα and eEF1Bβ proteins have structurally conserved GEF domains, their total number within the complex is equal to six. Such, so far, unique structural assembly of the guanine-nucleotide exchange factors within a stable complex may be considered as a ‘GEF hub’ that ensures efficient maintenance of the translationally active GTP-bound conformation of eEF1A in higher eukaryotes.

Structural analogue-based protein structure domain assembly assisted by deep learning.

With the breakthrough of AlphaFold2, the protein structure prediction problem has made remarkable progress through deep learning end-to-end techniques, in which correct folds could be built for nearly all single-domain proteins. However, the full-chain modelling appears to be lower on average accuracy than that for the constituent domains and requires higher demand on computing hardware, indicating the performance of full-chain modelling still needs to be improved. In this study, we investigate whether the predicted accuracy of the full-chain model can be further improved by domain assembly assisted by deep learning. In this article, we developed a structural analogue-based protein structure domain assembly method assisted by deep learning, named SADA. In SADA, a multi-domain protein structure database was constructed for the full-chain analogue detection using individual domain models. Starting from the initial model constructed from the analogue, the domain assembly simulation was performed to generate the full-chain model through a two-stage differential evolution algorithm guided by the energy function with an inter-residue distance potential predicted by deep learning. SADA was compared with the state-of-the-art domain assembly methods on 356 benchmark proteins, and the average TM-score of SADA models is 8.1% and 27.0% higher than that of DEMO and AIDA, respectively. We also assembled 293 human multi-domain proteins, where the average TM-score of the full-chain model after the assembly by SADA is 1.1% higher than that of the model by AlphaFold2. To conclude, we find that the domains often interact in the similar way in the quaternary orientations if the domains have similar tertiary structures. Furthermore, homologous templates and structural analogues are complementary for multi-domain protein full-chain modelling. http://zhanglab-bioinf.com/SADA. Supplementary data are available at Bioinformatics online.

I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction.

Most proteins in cells are composed of multiple folding units (or domains) to perform complex functions in a cooperative manner. Relative to the rapid progress in single-domain structure prediction, there are few effective tools available for multi-domain protein structure assembly, mainly due to the complexity of modeling multi-domain proteins, which involves higher degrees of freedom in domain-orientation space and various levels of continuous and discontinuous domain assembly and linker refinement. To meet the challenge and the high demand of the community, we developed I-TASSER-MTD to model the structures and functions of multi-domain proteins through a progressive protocol that combines sequence-based domain parsing, single-domain structure folding, inter-domain structure assembly and structure-based function annotation in a fully automated pipeline. Advanced deep-learning models have been incorporated into each of the steps to enhance both the domain modeling and inter-domain assembly accuracy. The protocol allows for the incorporation of experimental cross-linking data and cryo-electron microscopy density maps to guide the multi-domain structure assembly simulations. I-TASSER-MTD is built on I-TASSER but substantially extends its ability and accuracy in modeling large multi-domain protein structures and provides meaningful functional insights for the targets at both the domain- and full-chain levels from the amino acid sequence alone.

Novel DNM1L variants impair mitochondrial dynamics through divergent mechanisms.

Imbalances in mitochondrial and peroxisomal dynamics are associated with a spectrum of human neurological disorders. Mitochondrial and peroxisomal fission both involve dynamin-related protein 1 (DRP1) oligomerisation and membrane constriction, although the precise biophysical mechanisms by which distinct DRP1 variants affect the assembly and activity of different DRP1 domains remains largely unexplored. We analysed four unreported de novo heterozygous variants in the dynamin-1-like gene DNM1L affecting different highly conserved DRP1 domains, leading to developmental delay, seizures, hypotonia, and/or rare cardiac complications in infancy. Single-nucleotide DRP1 stalk domain variants were found to correlate with more severe clinical phenotypes, with in vitro recombinant human DRP1 mutants demonstrating greater impairments in protein oligomerisation, DRP1-peroxisomal recruitment, and both mitochondrial and peroxisomal hyperfusion compared to GTPase or GTPase-effector domain variants. Importantly, we identified a novel mechanism of pathogenesis, where a p.Arg710Gly variant uncouples DRP1 assembly from assembly-stimulated GTP hydrolysis, providing mechanistic insight into how assembly-state information is transmitted to the GTPase domain. Together, these data reveal that discrete, pathological DNM1L variants impair mitochondrial network maintenance by divergent mechanisms.

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Catalytic cycling of human mitochondrial Lon protease

The mitochondrial Lon protease (LonP1) regulates mitochondrial health by removing redundant proteins from the mitochondrial matrix. We determined LonP1 in eight nucleotide-dependent conformational states by cryoelectron microscopy (cryo-EM). The flexible assembly of N-terminal domains had 3-fold symmetry, and its orientation depended on the conformational state. We show that a conserved structural motif around T803 with a high similarity to the trypsin catalytic triad is essential for proteolysis. We show that LonP1 is not regulated by redox potential, despite the presence of two conserved cysteines at disulfide-bonding distance in its unfoldase core. Our data indicate how sequential ATP hydrolysis controls substrate protein translocation in a 6-fold binding change mechanism. Substrate protein translocation, rather than ATP hydrolysis, is a rate-limiting step, suggesting that LonP1 is a Brownian ratchet with ATP hydrolysis preventing translocation reversal. 3-fold rocking motions of the flexible N-domain assembly may assist thermal unfolding of the substrate protein.

Virus Hijacks Host Proteins and Machinery for Assembly and Budding, with HIV-1 as an Example.

Viral assembly and budding are the final steps and key determinants of the virus life cycle and are regulated by virus–host interaction. Several viruses are known to use their late assembly (L) domains to hijack host machinery and cellular adaptors to be used for the requirement of virus replication. The L domains are highly conserved short sequences whose mutation or deletion may lead to the accumulation of immature virions at the plasma membrane. The L domains were firstly identified within retroviral Gag polyprotein and later detected in structural proteins of many other enveloped RNA viruses. Here, we used HIV-1 as an example to describe how the HIV-1 virus hijacks ESCRT membrane fission machinery to facilitate virion assembly and release. We also introduce galectin-3, a chimera type of the galectin family that is up-regulated by HIV-1 during infection and further used to promote HIV-1 assembly and budding via the stabilization of Alix–Gag interaction. It is worth further dissecting the details and finetuning the regulatory mechanism, as well as identifying novel candidates involved in this final step of replication cycle.

Orchestrating ribosomal RNA folding during ribosome assembly.

Construction of the eukaryotic ribosome is a complex process in which a nascent ribosomal RNA (rRNA) emerging from RNA Polymerase I hierarchically folds into a native three-dimensional structure. Modular assembly of individual RNA domains through interactions with ribosomal proteins and a myriad of assembly factors permit efficient disentanglement of the error-prone RNA folding process. Following these dynamic events, long-range tertiary interactions are orchestrated to compact rRNA. A combination of genetic, biochemical, and structural studies is now providing clues into how a nascent rRNA is transformed into a functional ribosome with high precision. With this essay, we aim to draw attention to the poorly understood process of establishing correct RNA tertiary contacts during ribosome formation.

Trinity of G-tetrads and origin of translation

BackgroundThe RNA world hypothesis cannot address most of the questions of the origin of life without violating the continuity principle (small Darwinian steps without foresight and miracles). Moreover, the RNA world is an isolated system incapable of accommodating the genetic code and evolving into extant biochemistry. All these problems are rooted in the central assumption of the hypothesis: de novo appearance of the ribozymes, production of which represents a multistep reaction requiring the complementarity principle. Thus, even the basis of the RNA world is at odds with the continuity principle—it uses foresight (multistep reaction) and a miracle (complementarity principle). Can a three-dimensional (3D) architecture, capable of molecular recognition and catalysis, be formed in a single-step reaction without the complementarity or any other preexisting rules?HypothesisAt first glance, the above question sounds rhetoric since the complementarity principle is the essential feature of the RNA world; it turns an RNA polymer into a genetic material. Without it, the RNA world becomes as shapeless and unconvincing as other hypotheses based on the non-hereditary molecules (i.e., protein world). However, it was suggested recently that the quadruplexes could initiate life and take necessary evolutionary steps before the arrival of the complementarity rules. The hypothesis relies on the unique properties of guanines (Gs) to self-assemble into G-tetrads and efficiently polymerize without any external help or preexisting rules. Interestingly, polyG folds into an unusually stable and well-structured monomolecular architecture that uses the quadruplex domain (QD) assembly. The QD has a strictly defined zigzag-like building pattern to accommodate only three G-tetrads. Since both QD architecture and codon length are based on triplets, the inevitable question arises: are they related? Or could QD play the role of the early adapter and determine the codon length? The current paper is an attempt to answer this question.ConclusionWhile without translation apparatus most of the steps of the extant translation are physically impossible, the QD-mediated translation is sterically feasible and can be explained by physicochemical properties of the QD and the amino acids without violating the continuity principle. Astonishingly, the quadruplex world hypothesis can address all the shortcomings of the RNA world, including its most significant challenge—step-by-step evolution from the polymerization of the first polynucleotide to the extant biochemistry.

Modulation of assembly of TDP-43 low-complexity domain by heparin: From droplets to amyloid fibrils

TAR DNA-binding protein 43 (TDP-43) is an RNA-regulating protein that carries out many cellular functions through liquid-liquid phase separation (LLPS). The LLPS of TDP-43 is mediated by its C-terminal low-complexity domain (TDP43-LCD) corresponding to the region 267–414. In neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia, pathological inclusions of the TDP-43 are found that are rich in the C-terminal fragments of ∼25 and ∼35 kDa, of which TDP43-LCD is a part. Thus, understanding the assembly process of TDP43-LCD is essential, given its involvement in the formation of both functional liquid-like assemblies and solid- or gel-like pathological aggregates. Here, we show that the solution pH and salt modulate TDP43-LCD LLPS. A gradual reduction in the pH below its isoelectric point of 9.8 results in a monotonic decrease of TDP43-LCD LLPS due to charge-charge repulsion between monomers, while at pH 6 and below no LLPS was observed. The addition of heparin to TDP43-LCD solution at pH 6, at a 1:2 heparin-to-TDP43-LCD molar ratio, promotes TDP43-LCD LLPS, while at higher concentration, it disrupts LLPS through a reentrant phase transition. Upon incubation at pH 6, TDP43-LCD undergoes gelation without phase separation. However, in the reentrant regime in the presence of a high heparin concentration, it forms thick amyloid aggregates that are significantly more SDS resistant than the gel. The results indicate that the material nature of the TDP43-LCD assembly products can be modulated by heparin which is significant in the context of liquid-to-solid phase transition observed in TDP-43 proteinopathies. Our findings are also crucial in relation to similar transitions that could occur due to alteration in the molecular level interactions among various multivalent biomolecules involving other LCDs and RNAs.