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[Display omitted] • Computational design stabilized human and bovine serum albumins. • Designs express solubly in E. coli and exhibit up to 40 °C increased thermostability. • Some designs exhibit identical ligand binding properties. • Crystal structure confirms design accuracy. • Designs can be used in cell culture and in vitro applications. Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties. [ABSTRACT FROM AUTHOR]

Carnivorous plants represent a so far underexploited reservoir of novel proteases with potentially useful activities. Here we investigate 44 cysteine proteases from the Cape sundew, Drosera capensis, predicted from genomic DNA sequences. D. capensis has a large number of cysteine protease genes; analysis of their sequences reveals homologs of known plant proteases, some of which are predicted to have novel properties. Many functionally significant sequence and structural features are observed, including targeting signals and occluding loops. Several of the proteases contain a new type of granulin domain. Although active site residues are conserved, the sequence identity of these proteases to known proteins is moderate to low; therefore, comparative modeling with all-atom refinement and subsequent atomistic MD-simulation is used to predict their 3D structures. The structure prediction data, as well as analysis of protein structure networks, suggest multifarious variations on the papain-like cysteine protease structural theme. This in silico methodology provides a general framework for investigating a large pool of sequences that are potentially useful for biotechnology applications, enabling informed choices about which proteins to investigate in the laboratory.

Membrane proteins make up approximately one third of all proteins, and they play key roles in a plethora of physiological processes. However, membrane proteins make up less than 2% of experimentally determined structures, despite significant advances in structure determination methods, such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. One potential alternative means of structure elucidation is to combine computational methods with experimental EPR data. In 2011, Hirst and others introduced RosettaEPR and demonstrated that this approach could be successfully applied to fold soluble proteins. Furthermore, few computational methods for de novo folding of integral membrane proteins have been presented. In this work, we present RosettaTMH, a novel algorithm for structure prediction of helical membrane proteins. A benchmark set of 34 proteins, in which the proteins ranged in size from 91 to 565 residues, was used to compare RosettaTMH to Rosetta’s two existing membrane protein folding protocols: the published RosettaMembrane folding protocol (“MembraneAbinitio”) and folding from an extended chain (“ExtendedChain”). When EPR distance restraints are used, RosettaTMH+EPR outperforms ExtendedChain+EPR for 11 proteins, including the largest six proteins tested. RosettaTMH+EPR is capable of achieving native-like folds for 30 of 34 proteins tested, including receptors and transporters. For example, the average RMSD100SSE relative to the crystal structure for rhodopsin was 6.1 ± 0.4 Å and 6.5 ± 0.6 Å for the 449-residue nitric oxide reductase subunit B, where the standard deviation reflects variance in RMSD100SSE values across ten different EPR distance restraint sets. The addition of RosettaTMH and RosettaTMH+EPR to the Rosetta family of de novo folding methods broadens the scope of helical membrane proteins that can be accurately modeled with this software suite.

Cell signaling is a fundamental process for all living organisms. G protein-coupled receptors (GPCRs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric G proteins. In order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. By interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. To construct this mapping of pairwise interactions in silico, we analyzed the Rhodopsin GPCR coupled to a Gαi1β1γ1 heterotrimer. The structure of this G protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric Gα(GTP) state. From these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the ROSETTA modeling software suite. Here we disseminate a comprehensive survey of all critical interactions and create intra-protein network communication maps. These networks represent nodes of interaction necessary for G protein activation.

Rough set theory is a new mathematical approach to analyze the imperfect knowledge. It does not need any preliminary or additional information about data and provides efficient methods, algorithms and tools for finding hidden patterns in uncertain data. In this study, RST has been used to extract the rules from the data of energy audits of buildings. Since part of building energy audit data related to assessment of occupants comfort level and other data related to the technical analysis of the buildings so in this research, a decision attribute and eleven conditional attributes have been selected and rules inference have been done using ROSETTA software. Due to the different algorithms of data complement, discretization, reduction and rule generation, four rule models have been constructed based on the conditions of this study. Cross validation is used for evaluation of the model results. Finally the best model was chosen with fourteen rules and 99.8 percent of accuracy. The model demonstrate that the core attribute of buildings is "uncontrolled area of buildings". It means that if the value of this attribute is calculated the 14 rules can be used to accurately predict the level of employees comfort in the buildings.

Understanding and changing the energy consumption behavior requires extensive knowledge about the motives of behavior. In this research, Rough Set Theory is used to investigate the energy consumption behavior of employees in organizations. So, thirteen condition attributes and a decision attribute are selected and the decision system is created. Condition attributes include demographic, values, attitudes and organizational characteristics of employees and decision attribute relates to energy consumption behavior. 482 employees are selected randomly from 37 office buildings of ministry of Petroleum and rough modeling are performed for them. By combining different methods of discretizing, reduction algorithms and rule generating, nine models are made using ROSETTA software. The results show that four of the 13 condition attributes, involving “organizational citizenship”, “satisfaction”, “attitude toward behavior” and “lighting control” are selected as the main features of the system. After cross validation of the various models, the model of manually discretizing using genetic algorithms and ORR approach to extract reducts has the most accuracy and selected as the most reliable model.

Rosetta Promontory, Egypt has been suffering from a continuous erosion problem. The dramatic retreatment was observed during the last century. It is basically due to the construction of Aswan High Dam in 1964, which reduced the flow and sediment discharges. In this paper, four Landsat images (two Thematic Mapper and two Enhanced Thematic Mapper) covering the period from 1984 to 2014 were used. These Landsat images were radio-metrically and geometrically corrected, and then, multi-temporal post-classification analysis was performed to detect land cover changes, extracting shoreline positions to estimate shoreline change rates of the Nile delta coast around Rosetta Promontory. This method provides a viable means for examining long-term shoreline changes. Four categories, including seawater, developed (agriculture and urban), sabkhas (salt-flat), and undeveloped areas, were selected to evaluate their temporal changes by comparing the four selected images. Supervised classification technique was used with support vector machine algorithm to detect temporal changes. The overall accuracy assessment of this method ranged from 97% to 100%. In addition, the shoreline was extracted by applying two different techniques. The first method is based on a histogram threshold of Band 5, and the other uses the combination of histogram threshold of Band 5 and two band ratios (Band 2/Band 4 and Band 2/Band 5). For land cover change detection from 1984 to 2014, it was found that the developed area that increased by 9% although the land in the study area has been contracted by 1.6% due to coastal erosion. The shoreline retreat rate has decreased more than 70% from 1984 to 2014. Nevertheless, it still suffers from significant erosion with a maximum rate of 37 m/year. In comparison to ground survey and different remote sensing techniques, the established trend of shoreline change extracted using histogram threshold was found to be closely consistent with these studies rather than combining band ratio with histogram threshold.

Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor's outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein-protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host-virus compatibility or therapeutic strategies based on the transferrin receptor decoys., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Abstract The international Rosetta mission was launched on 2nd March 2004 for a ten year journey to its target destination, comet 67P/Churyumov-Gerasimenko. Following the January 2014 exit from a 31 month hibernation period, Rosetta approached and rendezvoused with the comet in August 2014. On 12th November 2014, the Philae lander was deployed from Rosetta onto the comet's surface after which the Orbiter continued its comet escort phase. The Rosetta Science Ground Segment supports the Project Scientist and the Science Working Team, in order to ensure the coordination, development, validation and delivery of the desired science operations plans and their associated operational products throughout the mission, and to ensure the preservation of adequate data to the Planetary Science Archive. Within the Science Ground Segment, the Operations and Planning Group is responsible for the medium, short and very short term cycles, a stepped sequential process that implements the higher level science long term plan into the final science pointing requests and the command sequences to be executed on board the spacecraft. In this paper we will present the Operations and Planning group and its science planning activities, focusing on the evolution of the Science Operations implementation through the Rosetta mission phases. We will describe the day-to-day work needed to achieve science operations around the challenging and previously unknown cometary environment, and the software tools that support the planning process. We will finally outline several conclusions after the experience gained in the ∼2 years of operations at the comet. Highlights • Space science operations discipline combines scientific and engineering knowledge. • Rosetta successfully returned unique science results in its two years at comet 67P. • Rosetta changed from a rigid long-lead concept to a close-to-execution agile planning. • Rosetta integrated new planning rules in the harsh fickle comet environment. • It is remarkable how fast planning was adapted while performing comet observations. [ABSTRACT FROM AUTHOR]

Abstract Protein tertiary structure modeling is a critical step for the interpretation of three dimensional (3D) election microscopy density. Our group participated the 2015/2016 EM Model Challenge using the MAINMAST software for a de novo main chain modeling. The software generates local dense points using the mean shifting algorithm, and connects them into Cα models by calculating the minimum spanning tree and the longest path. Subsequently, full atom structure models are generated, which are subject to structural refinement. Here, we summarize the qualities of our submitted models and examine successful and unsuccessful models, including 3D models we did not submit to the Challenge. Our protocol using the MAINMAST software was sometimes able to build correct conformations with 3.4–5.1 Å RMSD. Unsuccessful models had failure of chain traces, however, their Cα positions and some local structures were quite correctly built. For evaluate the quality of the models, the MAINMAST software provides a confidence score for each Cα position from the consensus of top 100 scoring models. [ABSTRACT FROM AUTHOR]

Zusammenfassung Der mit dem Instrument COSIMA gesammelte Staub des Kometen 67P/Tschurjumow‐Gerassimenko ist porös und besteht aus 10 bis 40 µm großen Agglomeraten. Van‐der‐Waals‐Kräfte halten diese zusammen. Die Teilchen bestehen zu einem Drittel aus Kohlenstoff. Vergleichbare hochporöse Staubteilchen haben wir bisher auf der Erde nicht gefunden, wenn auch eine Reihe von Meteoriten und Mikrometeoriten oder in der Atmosphäre aufgesammelte interplanetare Staubteilchen einzelne Eigenschaften der mit Rosetta gemachten Beobachtungen gut widerspiegeln. Zwei Jahre lang begleitete die Raumsonde Rosetta den Kometen Tschurjumow‐Gerassimenko. Währenddessen untersuchte das an Bord befindliche Instrument COSIMA von ihm abströmende Staubteilchen und ermittelte deren mechanische, elektrische und optische Eigenschaften sowie die chemische Zusammensetzung. [ABSTRACT FROM AUTHOR]

Background: With increasing interest in ab initio protein design, there is a desire to be able to fully explore the design space of insertions and deletions. Nature inserts and deletes residues to optimize energy and function, but allowing variable length indels in the context of an interactive protein design session presents challenges with regard to speed and accuracy. Results: Here we present a new module (INDEL) for InteractiveRosetta which allows the user to specify a range of lengths for a desired indel, and which returns a set of low energy backbones in a matter of seconds. To make the loop search fast, loop anchor points are geometrically hashed using C α-C α and C β-C β distances, and the hash is mapped to start and end points in a pre-compiled random access file of non-redundant, protein backbone coordinates. Loops with superposable anchors are filtered for collisions and returned to InteractiveRosetta as poly-alanine for display and selective incorporation into the design template. Sidechains can then be added using RosettaDesign tools. Conclusions: INDEL was able to find viable loops in 100% of 500 attempts for all lengths from 3 to 20 residues. INDEL has been applied to the task of designing a domain-swapping loop for T7-endonuclease I, changing its specificity from Holliday junctions to paranemic crossover (PX) DNA. [ABSTRACT FROM AUTHOR]

The Optical, Spectroscopic, and Infrared Remote Imaging System (OSIRIS) onboard the European Space Agency's Rosetta spacecraft acquired images of comet 67P/Churyumov–Gerasimenko (67P) and its surrounding dust coma starting from May 2014 until September 2016. In this paper we present methods and results from analysis of OSIRIS images regarding the dust outflow in the innermost coma of 67P. The aim is to determine the global dust outflow behaviour and place constraints on physical processes affecting particles in the inner coma. We study the coma region right above the nucleus surface, spanning from the nucleus centre out to a distance of about 50 km comet centric distance (approximately 25 average comet radii). We primarily adopt an approach used by Thomas and Keller (1990) to study the dust outflow. We present the effects on azimuthally-averaged values of the dust reflectance of non-radial flow and non-point-source geometry, acceleration of dust particles, sublimation of icy dust particles after ejection from the surface, dust particle fragmentation, optical depth effects and the influence of gravitationally bound particles. All of these physical processes could modify the observed distribution of light scattered by the dust coma. In the image analysis, profiles of azimuthally averaged dust brightness as a function of impact parameter b (azimuthal average, “Ā-curve”) were fitted with a simple function that best fits the shape of our profile curves ( f ( b ; u , v , w , z ) = u / b v + w b + z ). The analytical fit parameters ( u, v, w, z ), which hold the key information about the dust outflow behaviour, were saved in a comprehensive database. Through statistical analysis of these information, we show that the spatial distribution of dust follows free-radial outflow behaviour (i.e. force-free radial outflow with constant velocity) beyond distances larger than ∼11.9 km from the comet centre, which corresponds to a relative distance of about 6 average comet radii from the comet centre. Hence, we conclude that beyond this distance, and on average, fragmentation and gravitationally bound particles are negligible processes in determining the optically scattered light distribution in the innermost coma. Closer to the nucleus we observe dust outflow behaviour that deviates from free-radial outflow. A comparison of our result profiles with numerical models using a Direct Simulation Monte Carlo (DSMC) approach with dust particle distributions calculated using a test particle approach has been used to demonstrate the influence of a complex shape and particle acceleration on the azimuthal average profiles. We demonstrate that, while other effects such as fragmentation or sublimation of dust particles cannot be ruled out, acceleration of the dust particles and effects arising from the shape of the irregular nucleus (non-point source geometry) are sufficient to explain the observed dust outflow behaviour from image data analysis. As a by-product of this work, we have calculated “Afρ” values for the 1/r regime. We found a peak in the coma activity in terms of Afρ (normalised to a phase angle of 90°) of ∼210 cm 20 days after perihelion. Furthermore, based on simplified models of particle motion within bound orbits, it is shown that limits on the total cross-sectional area of bound particles might be derived through further analysis. An example is given. [ABSTRACT FROM AUTHOR]

Disulfide‐rich peptides represent an important protein family with broad pharmacological potential. Recent advances in computational methods have made it possible to design new peptides which adopt a stable conformation de novo. Here, we describe a system to produce disulfide‐rich de novo peptides using Escherichia coli as the expression host. The advantage of this system is that it enables production of uniformly 13C‐ and 15N‐labeled peptides for solution nuclear magnetic resonance (NMR) studies. This expression system was used to isotopically label two previously reported de novo designed peptides, and to determine their solution structures using NMR. The ensemble of NMR structures calculated for both peptides agreed well with the design models, further confirming the accuracy of the design protocol. Collection of NMR data on the peptides under reducing conditions revealed a dependency on disulfide bonds to maintain stability. Furthermore, we performed long‐time molecular dynamics (MD) simulations with tempering to assess the stability of two families of de novo designed peptides. Initial designs which exhibited a stable structure during simulations were more likely to adopt a stable structure in vitro, but attempts to utilize this method to redesign unstable peptides to fold into a stable state were unsuccessful. Further work is therefore needed to assess the utility of MD simulation techniques for de novo protein design. [ABSTRACT FROM AUTHOR]

Designed helical repeats (DHRs) are modular helix-loop-helix-loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions. [ABSTRACT FROM AUTHOR]

Computational protein design is a set of procedures for computing amino acid sequences that will fold into a specified structure. Rosetta Design, a commonly used software for protein design, allows for the effective identification of sequences compatible with a given backbone structure, while molecular dynamics (MD) simulations can thoroughly sample near-native conformations. We benchmarked a procedure in which Rosetta design is started on MD-derived structural ensembles and showed that such a combined approach generates 20–30% more diverse sequences than currently available methods with only a slight increase in computation time. Importantly, the increase in diversity is achieved without a loss in the quality of the designed sequences assessed by their resemblance to natural sequences. We demonstrate that the MD-based procedure is also applicable to de novo design tasks started from backbone structures without any sequence information. In addition, we implemented a protocol that can be used to assess the stability of designed models and to select the best candidates for experimental validation. In sum our results demonstrate that the MD ensemble-based flexible backbone design can be a viable method for protein design, especially for tasks that require a large pool of diverse sequences. [ABSTRACT FROM AUTHOR]

Abstract: The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs‐into‐holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes. [ABSTRACT FROM AUTHOR]

Abstract: A major point of interest in cometary plasma physics has been the diamagnetic cavity, an unmagnetized region in the innermost part of the coma. Here we combine Langmuir and Mutual Impedance Probe measurements to investigate ion velocities and electron temperatures in the diamagnetic cavity of comet 67P, probed by the Rosetta spacecraft. We find ion velocities generally in the range 2–4 km/s, significantly above the expected neutral velocity ≲1 km/s, showing that the ions are (partially) decoupled from the neutrals, indicating that ion‐neutral drag was not responsible for balancing the outside magnetic pressure. Observations of clear wake effects on one of the Langmuir probes showed that the ion flow was close to radial and supersonic, at least with respect to the perpendicular temperature, inside the cavity and possibly in the surrounding region as well. We observed spacecraft potentials ≲ − 5 V throughout the cavity, showing that a population of warm (∼5 eV) electrons was present throughout the parts of the cavity reached by Rosetta. Also, a population of cold ( ≲ 0 . 1 eV) electrons was consistently observed throughout the cavity, but less consistently in the surrounding region, suggesting that while Rosetta never entered a region of collisionally coupled electrons, such a region was possibly not far away during the cavity crossings. [ABSTRACT FROM AUTHOR]

Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amounts, thus hampering research and applications. Research has revealed the thermodynamic, cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic analysis with atomistic design, have shown drastic improvements in solubility, thermal stability, and aggregation resistance while maintaining the protein's primary molecular activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodology to large proteins and molecular activities that have proven challenging in the past. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the distribution pattern of persistent organic pollutants in water, sediment and aquatic biota represented by Oreochromis niloticus and Donax trunculus at the Rosetta Nile branch estuary. α-HCH, p,p′-DDE and polychlorinated biphenyls were the predominant compounds detected at ranges of 0.54–4.90 ng/l water, 0.75–2.41 ng/g, d. wt. sediment and 2.19–28.11 ng/g, fresh wt. biota. β and γ-HCHs, endosulfan compounds, heptachlor and heptachlor epoxide were at low detection frequencies. Totally, the organochlorine pollutants were at high levels and abundances in Donax spp. than in Tilapia spp. followed by sediment and water. These levels were ranged between lower and higher than those found by the other studies established in Egypt, and well below its tolerable residue levels in fish. A correlation was found for the quantified pollutants between water, sediment and biota. This is clearly reflecting the bioaccumulation properties of these compounds. [ABSTRACT FROM AUTHOR]

Understanding the function of high-density lipoprotein (HDL) requires detailed knowledge of the structure of its primary protein, apolipoprotein A-I (APOA1). However, APOA1 flexibility and HDL heterogeneity have confounded decades of efforts to determine high-resolution structures and consistent models. Here, molecular dynamics simulations totaling 30 μs on two nascent HDLs, each with 2 APOA1 and either 160 phospholipids and 24 cholesterols or 200 phospholipids and 20 cholesterols, show that residues 1-21 of the Nterminal domains of APOA1 interact via strong salt bridges. Residues 26-43 of one APOA1 in the smaller particle form a hinge on the disc edge, which displaces the C-terminal domain of the other APOA1 to the phospholipid surface. The proposed structures are supported by chemical cross-linking, Rosetta modeling of the N-terminal domain, and analysis of the lipid-free Δ185APOA1 crystal structure. These structures provide a framework for understanding HDL maturation and revise all previous models of nascent HDL. [ABSTRACT FROM AUTHOR]

Abstract: The instrument Cometary Secondary Ion Mass Analyzer (COSIMA) on board of the European Space Agency mission Rosetta to the comet 67P/Churyumov‐Gerasimenko is a secondary ion mass spectrometer with a time‐of‐flight mass analyzer. It collected near the comet several thousand particles, imaged them, and analyzed the elemental and chemical compositions of their surfaces. In this study, variables have been generated from the spectral data covering the mass ranges of potential C‐, H‐, N‐, and O‐containing ions. The variable importance in binary discriminations between spectra measured on cometary particles and those measured on the target background has been estimated by the univariate t test and the multivariate methods discriminant partial least squares, random forest, and a robust method based on the log ratios of all variable pairs. The results confirm the presence of organic substances in cometary matter—probably a complex macromolecular mixture. [ABSTRACT FROM AUTHOR]

Computing conformations which are essential to associate structural and functional information with gene sequences, is challenging due to the high dimensionality and rugged energy surface of the protein conformational space. Consequently, the dimension of the protein conformational space should be reduced to a proper level, and an effective exploring algorithm should be proposed. In this paper, a plug-in method for guiding exploration in conformational feature space with Lipschitz underestimation (LUE) for ab-initio protein structure prediction is proposed. The conformational space is converted into ultrafast shape recognition (USR) feature space firstly. Based on the USR feature space, the conformational space can be further converted into Underestimation space according to Lipschitz estimation theory for guiding exploration. As a consequence of the use of underestimation model, the tight lower bound estimate information can be used for exploration guidance, the invalid sampling areas can be eliminated in advance, and the number of energy function evaluations can be reduced. The proposed method provides a novel technique to solve the exploring problem of protein conformational space. LUE is applied to differential evolution (DE) algorithm, and metropolis Monte Carlo(MMC) algorithm which is available in the Rosetta; When LUE is applied to DE and MMC, it will be screened by the underestimation method prior to energy calculation and selection. Further, LUE is compared with DE and MMC by testing on 15 small-to-medium structurally diverse proteins. Test results show that near-native protein structures with higher accuracy can be obtained more rapidly and efficiently with the use of LUE. [ABSTRACT FROM AUTHOR]

Abstract: We describe several notable aspects of our structure predictions using Rosetta in CASP12 in the free modeling (FM) and refinement (TR) categories. First, we had previously generated (and published) models for most large protein families lacking experimentally determined structures using Rosetta guided by co‐evolution based contact predictions, and for several targets these models proved better starting points for comparative modeling than any known crystal structure—our model database thus starts to fulfill one of the goals of the original protein structure initiative. Second, while our “human” group simply submitted ROBETTA models for most targets, for six targets expert intervention improved predictions considerably; the largest improvement was for T0886 where we correctly parsed two discontinuous domains guided by predicted contact maps to accurately identify a structural homolog of the same fold. Third, Rosetta all atom refinement followed by MD simulations led to consistent but small improvements when starting models were close to the native structure, and larger but less consistent improvements when starting models were further away. [ABSTRACT FROM AUTHOR]

Abstract: Many naturally occurring protein systems function primarily as symmetric assemblies. Prediction of the quaternary structure of these assemblies is an important biological problem. This article describes automated tools we have developed for predicting the structures of symmetric protein assemblies in the Robetta structure prediction server. We assess the performance of this pipeline on a set of targets from the recent CASP12/CAPRI blind quaternary structure prediction experiment. Our approach successfully predicted 5 of 7 symmetric assemblies in this challenge, and was assessed as the best participating server group, and 1 of only 2 groups (human or server) with 2 predictions judged as high quality by the assessors. We also assess the method on a broader set of 22 natively symmetric CASP12 targets, where we show that oligomeric modeling can improve the accuracy of monomeric structure determination, particularly in highly intertwined oligomers. [ABSTRACT FROM AUTHOR]

Replacement of the sulfur atom in biologically active diaryl and heteroaryl thioethers (Ar–S–Ar’, HAr–S–Ar, and HAr–S–HAr’) with any of several one-atom or two-atom linkers can be expected to reduce the susceptibility of the analogue to metabolic oxidation, a well-documented problem for thioethers intended for medicinal chemistry applications. Ab initio calculations indicate how well various proposed thioether isosteric groups, including some new and unusual ones, may perform structurally and electronically in replacing the bridging sulfur atom. Four of these are calculationally evaluated as proposed substructures in Axitinib analogues. The predicted binding behavior of the latter within two different previously crystallographically characterized protein-Axitinib binding sites (VEGFR2 kinase and ABL1 T315I gatekeeper mutant kinase), and an assessment of their suitability and anticipated shortcomings, are presented. [ABSTRACT FROM AUTHOR]

On 12 November 2014, during the descent of the Rosetta lander Philae to the surface of comet 67P/Churyumov–Gerasimenko the Dust Impact Monitor (DIM) on board Philae recorded an impact of a cometary dust impact of a cometary dust particle at 2.4 km from the comet surface (5 km from the nucleus’ barycentre). In this work, we report further experiments that support the identification of this particle. We use aerogel as a comet analog material to characterise the properties of this particle. Our experiments show that this particle has a radius of 0.9 mm, a low density of 0.25 g/cm 3 and a high porosity close to 90%. The particle likely moved at near 4 m/s with respect to the comet. [ABSTRACT FROM AUTHOR]

ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa. The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein-protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron-electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest- energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins. [ABSTRACT FROM AUTHOR]

The Rosetta spacecraft was launched in 2004 and, after several planetary and two asteroid fly-bys, arrived at comet 67P/Churyumov-Gerasimenko in August 2014. After escorting the comet for two years and executing its scientific observations, the mission ended on 30 September 2016 through a touch down on the comet surface. This paper describes how the Planetary Science Archive (PSA) and the Planetary Data System – Small Bodies Node (PDS-SBN) worked with the Rosetta instrument teams to prepare the science data collected over the course of the Rosetta mission for inclusion in the science archive. As Rosetta is an international mission in collaboration between ESA and NASA, all science data from the mission are fully archived within both the PSA and the PDS. The Rosetta archiving process, supporting tools, archiving systems, and their evolution throughout the mission are described, along with a discussion of a number of the challenges faced during the Rosetta implementation. The paper then presents the current status of the archive for each of the science instruments, before looking to the improvements planned both for the archive itself and for the Rosetta data content. The lessons learned from the first 13 years of archiving on Rosetta are finally discussed with an aim to help future missions plan and implement their science archives. [ABSTRACT FROM AUTHOR]

Ensembles of protein structures are increasingly used to represent the conformational variation of a protein as determined by experiment and/or by molecular simulations, as well as uncertainties that may be associated with structure determinations or predictions. Making the best use of such information requires the ability to quantitatively compare entire ensembles. For this reason, we recently introduced the Ensemblator (Clark et al., Protein Sci 2015; 24:1528), a novel approach to compare user-defined groups of models, in residue level detail. Here we describe Ensemblator v3, an open-source program that employs the same basic ensemble comparison strategy but includes major advances that make it more robust, powerful, and user-friendly. Ensemblator v3 carries out multiple sequence alignments to facilitate the generation of ensembles from non-identical input structures, automatically optimizes the key global overlay parameter, optionally performs 'ensemble clustering' to classify the models into subgroups, and calculates a novel 'discrimination index' that quantifies similarities and differences, at residue or atom level, between each pair of subgroups. The clustering and automatic options mean that no pre-knowledge about an ensemble is required for its analysis. After describing the novel features of Ensemblator v3, we demonstrate its utility using three case studies that illustrate the ease with which complex analyses are accomplished, and the kinds of insights derived from clustering into subgroups and from the detailed information that locates significant differences. The Ensemblator v3 enhances the structural biology toolbox by greatly expanding the kinds of problems to which this ensemble comparison strategy can be applied. [ABSTRACT FROM AUTHOR]

Computational membrane protein design is challenging due to the small number of high-resolution structures available to elucidate the physical basis of membrane protein structure, multiple functionally important conformational states, and a limited number of high-throughput biophysical assays to monitor function. However, structural determination of membrane proteins has made tremendous progress in the past years. Concurrently the field of soluble computational design has made impressive inroads. These developments allow us to tackle the formidable challenge of designing functional membrane proteins. Herein, Rosetta is benchmarked for membrane protein design. We evaluate strategies to cope with the often reduced quality of experimental membrane protein structures. Further, we test the usage of symmetry in design protocols, which is particularly important as many membrane proteins exist as homo-oligomers. We compare a soluble scoring function with a scoring function optimized for membrane proteins, RosettaMembrane. Both scoring functions recovered around half of the native sequence when completely redesigning membrane proteins. However, RosettaMembrane recovered the most native-like amino acid property composition. While leucine was overrepresented in the inner and outer-hydrophobic regions of RosettaMembrane designs, it resulted in a native-like surface hydrophobicity indicating that it is currently the best option for designing membrane proteins with Rosetta. [ABSTRACT FROM AUTHOR]

Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders. [Display omitted] • Structures of peptide transporter DtpB in complex with 14 different di- and tripeptides • Binding affinities for more than 80 peptides based on thermal shifts • High-affinity peptides are poorly transported • Molecular docking of all 8,400 possible di- and tripeptides into DtpB Kotov et al. determine crystal structures of the peptide transporter DtpB bound to 14 different peptides. This work provides novel insights into the plasticity of the conserved binding pocket. The authors deploy state-of-the-art molecular docking and machine learning to probe whether all 8,400 possible di- and tripeptides bind to DtpB. [ABSTRACT FROM AUTHOR]

In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially due to the lack of comprehensive datasets including a structural readout. In addition, the recent breakthrough in structure prediction through deep learning demands an update of computational deletion mutation prediction. In this study, we deleted individually every residue of a small α-helical sterile alpha motif domain and investigated the structural and thermodynamic changes using 2D NMR spectroscopy and differential scanning fluorimetry. Then, we tested computational protocols to model and classify observed deletion mutants. We show a method using AlphaFold2 followed by RosettaRelax performs the best overall. In addition, a metric containing pLDDT values and Rosetta ΔΔG is most reliable in classifying tolerated deletion mutations. We further test this method on other datasets and show they hold for proteins known to harbor disease-causing deletion mutations. [Display omitted] • Experimental investigation of in-frame deletion mutations in an α-helical protein • Biomolecular NMR and stability tests to characterize tolerated deletion mutations • Comparison of computational protocols to model deletion mutations • A combination of Rosetta ΔΔGs and AlphaFold2 pLDDT predicts solubility We lack datasets to develop computational tools that can help predict the effect of in-frame deletion mutations on protein structure and function. Woods et al. present a comprehensive structural and biophysical analysis of a series of deletion mutations. The experimental results are evaluated against computational predictions using AlphaFold2 and Rosetta. [ABSTRACT FROM AUTHOR]