Mutations in the N-terminal peptide (Ser-Thr to Ala-Gly substitution) of the coat protein (CP) of potato virus X (PVX-ST) render its genomic RNA translationally competent, unlike in the wild-type PVX virions. Consequently, RNA within the PVX-ST virions can be translated without additional triggers (such as phosphorylation or interaction with the triple gene block 1 protein), unlike the encapsidated RNA of the wild-type virus. Comprehensive structural analysis using molecular dynamics (MD), small-angle X-ray scattering (SAXS), and tritium planigraphy revealed differences in the virion organization. The mutations were shown to increase hydrophobicity and induce partial folding of the N-terminal peptides. This triggers structural rearrangement in the PVX-ST virion: packing density of the coat proteins within the helical capsid is altered. This conclusion is supported by the SAXS data, increased accessibility for tritium labeling of the key CP domains (including the RNA-binding region), and reduced stability against the action of the sodium dodecyl sulfate detergent. The obtained results provide explanation for the mechanism by which the encapsidated RNA of the PVX-ST mutant becomes accessible to ribosomes. This mechanism is associated with structural rearrangement of the N-terminal coat protein peptide and change in the packing density of the helical capsid.

Capsids of the potexvirus family virions are characterized by the presence on the surface of virions of partially disordered N-terminal protein fragments of various lengths. The present study is devoted to studying the effect of in situ removal of the N-terminal domain of coat protein (CP) on structural organization and physicochemical properties of the potato virus X (PVX) virions. The work considers PVX virions containing an intact Ps-form CP, as well as virions including an in situ degraded Pf-form devoid of 19/21 amino acid residues from the N-end (PVXΔN). Synchrotron small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), tritium bombardment, and several other physicochemical methods were used in the study. Analysis of the images obtained using TEM revealed similarities in the architecture of filamentous PVX and PVXΔN virions. SAXS results demonstrated differences in organization of the capsid of PVX and PVXΔN virions: the latter was characterized by the reduced size of the ordered regions, indicating partial disruption of the structure of the viral protein framework. In addition, based on the SAXS scattering curves, parameters of the spiral packing of virions in solution were calculated, and structural modeling of particles was performed. Modeling results also indicate changes in the structure of the capsid due to removal of the ΔN-peptide. Using information about the secondary structure of the PVX model (PDB ID: 6R7G) and data from our previous studies on tritium labeling of the surface sites of PVX and PVXΔN virions, comparative analysis of the label incorporation profiles into elements of the protein secondary structure was conducted. This approach made it possible to predict localization of the ΔN-peptide above the amino acid residues of neighboring helical subunits (122-129 and 143-153) and demonstrate stabilizing role of this peptide on the overall structure of the virion. Increase in the level of labelling in the C-terminal region after removal of the ΔN-peptide also indicates decrease in the compactness of the virion. In general, the gained knowledge could be useful when using virus-like nanoparticles in biotechnology.

The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

The results of investigation of DNA-protein complexes by two independent structural methods, namely, molecular dynamics (MD) and small-angle X-ray scattering (SAXS), were compared. Molecular dynamics is a computational method enabling the visualization of the behavior of macromolecules in real media, which is based on the laws of physics, but it is limited by numerous simplifications. Small-angle X-ray scattering is an X-ray method, which allows the reconstruction of the three-dimensional structure of a system in solution from a one-dimensional small-angle scattering profile, but it faces the problem of ambiguity in solving inverse problems. The use of structural characteristics of the complexes determined by SAXS for the validation of 3D structural models generated by MD simulations made it possible to significantly reduce the ambivalence of theoretical predictions and demonstrated the efficiency of a combination of MD simulations and SAXS for solving problems of structural biology.

The oligomeric state of the nucleoid-associated protein IHF (integration host factor) plays a significant role in the organization and compaction of the bacterial nucleoid and also in the evolution of bacterial resistance to unfavorable environmental conditions, in particular to antibiotics. Although IHF was identified more than 25 years ago, the molecular mechanisms of its participation in these processes are poorly understood. Using small-angle X-ray scattering, it was demonstrated for the first time that there are different oligomeric states of IHF in an aqueous medium depending on the presence of metal cations. It was found that the presence of Mg2+ and K+ ions hinders the formation of high-order oligomers of IHF. The results of this study may be useful in the development of strategies against bacterial resistance to drugs.

Biomolecular corona is the major obstacle to the clinical translation of nanomedicines. Since corona formation is governed by molecular interactions at the nano-bio interface, nanoparticle surface properties such as topography, charge and surface chemistry can be tuned to manipulate biomolecular corona formation. To this end, as the first step towards a deep understanding of the processes of corona formation, it is necessary to develop nanoparticles employing various biocompatible materials and characterize their surface structure and dynamics at the molecular level. In this work, we applied molecular dynamics simulation to study the surface structure of organic core-shell nanoparticles formed by the self-assembly of synthetic molecules composed of a DOPE lipid, a carboxymethylglycine spacer and biotin. Lipid moieties form the hydrophobic core, spacer motifs serve as a hydrophilic shell and biotin residues function as a targeting ligand. By mixing such function-spacer-lipid, spacer-lipid and lipid-only constructs at various molar ratios, densities of the ligand and spacer on the nanoparticle surface were modified. For convenient analysis of the structure and dynamics of all regions of the nanoparticle surface, we compiled topography maps based on atomic coordinates. It was shown that an increase in the density of the shell does not reduce exposure of the core, but increases shell average thickness. Biotin, due to its alkyl valeric acid chain and spacer flexibility, is localized primarily near the hydrophobic core and its partial presentation on the surface occurs only in nanoparticles with higher ligand densities. However, an increase in biotin density leads to its clustering. In turn, ligand clustering diminishes the stealth properties of the shell and targeting efficiency. Based on nanoparticle surface structures, we determined the optimal density of biotin. Experimental studies reported in the literature confirm these conclusions. We also suggest design tips to achieve the preferred biotin presentation. Simulation results are consistent with the synchrotron SAXS profile. We believe that such studies will contribute to a better understanding of nano-bio interactions towards the rational design of efficient drug delivery systems.

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share 40

The amino acid sequences of coat proteins (CPs) of potexviruses such as potato virus X (PVX) and alternanthera mosaic virus (AltMV) share about 40% sequence identity. However, the N-terminal CP domains of these virions differ both in length (the N-terminal CP domain of PVX is longer by 28 residues, ∆N = 28), and in amino acid sequence. In this work, we determined the effect of the N-terminal CP domain on the structure and physicochemical properties of the entire PVX and AltMV virions. It was shown that the melting point of PVX samples is 10-12°C higher than that of AltMV preparations; the circular dichroism spectra of these viruses also differ significantly. Spatial alignment of the existing high-resolution potexvirus CP structures showed that the RMSD value between Cα-atoms was the largest for the N-terminal domains of the two compared models. From computer simulations the ∆N-terminal CP domain of PVX is completely disordered. According to synchrotron small-angle X-ray scattering (SAXS) data, the structure of CP of PVX and AltMV virions differs, in particular, CP PVX has a larger size of crystallinity regions and, therefore, is more ordered. Using SAXS, virion diameters and helix parameters in solution are calculated. The influence of the conformation and localization of the N-terminal domain of PVX CP relative to the surface of the virion on its structure was revealed. Presumably, the increased thermal stability of PVX virions compared to AltMV is provided by the elongated N-terminal domains (ΔN = 28), which ensures additional contact between the adjacent CP subunits in the PVX virion.

For the biomedical applications of nanoparticles, the study of their structure is a major step towards understanding the mechanisms of their interaction with biological environment. Detailed structural analysis of particles' surface is vital for rational design of drug delivery systems. In particular, for core-shell or surface-modified nanoparticles surface structure can be described in terms of shell coating uniformity and shell thickness uniformity around the nanoparticle core. Taken together, these terms can be used to indicate degree of heterogeneity of nanoparticle surface structure. However, characterization of nanoparticle surface structure under physiological conditions is challenging due to limitations of experimental techniques. In this paper, we apply SAXS contrast variation combined with ab initio bead modeling for this purpose. Approach is based on the fact that nanoparticles under study are produced by self-assembly of phospholipid-conjugated molecules that possess moieties with significantly different electron densities enabling SAXS technique to be used to distinguish nanoparticle shell and study its structure. Ab initio single phase and ab initio multiphase modeling based on SAXS curve of nanoparticles in phosphate buffer solution allowed to reconstruct nanoparticle shell coating and assess its uniformity, while serial nanoparticle reconstructions from solutions with gradually increased solvent electron densities revealed relative shell coating thickness around nanoparticle core. Nanoparticle shell structure representation was verified by molecular dynamics simulation and derived full-atom nanoparticle shell structure showed good agreement with SAXS-derived representation. Obtained data indicate that studied nanoparticles exhibit highly heterogeneous surface structure.

The structures of hydrophobic membrane proteins are studied using matrices, which serve as models of cell membranes and are formed by the appropriate amphiphilic molecules, e.g., by surfactant or lipid molecules. To study the structure of a protein incorporated into an artificial membrane, first of all it is necessary to determine the structure of the membrane. The ELLLIP and ELLMIC algorithms were previously developed to address this issue by small-angle X-ray scattering. These algorithms allow the construction of models of ellipsoidal vesicles based on the atomic structure of a lipid or surfactant monomer. However, the results of modeling depend, to a large extent, on the subjective assessment of the initial values of the structural parameters of the matrices and may be wrong due to the ambiguity in the solution of such problems. Here, we present an independent approach to the determination of the initial sizes of model membranes for their subsequent structural modeling, which is based on the analysis of the pair-distance distribution functions derived directly from the small-angle X-ray scattering curve.

Structure and function of bacterial nucleoid is controlled by the nucleoid-associated proteins (NAP). In any phase of growth, various NAPs, acting sequentially, condense nucleoid and facilitate formation of its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, Dps protein, is strongly expressed, and DNA–protein crystals are formed that transform nucleoid into a static, transcriptionally inactive structure, effectively protected from the external influences. Discovery of crystal structures in living cells and association of this phenomenon with the bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes formation of the protective DNA–Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying structure of proteins in solution, and dynamic light scattering as a complementary one. To interpret the SAXS data, various approaches and computer programs were used (in particular, the evaluation of structural invariants, rigid body modeling and equilibrium mixture analysis in terms of the volume fractions of its components were applied), which made it possible to determine macromolecular characteristics and obtain reliable 3D structural models of various oligomeric forms of HU and IHF proteins with 2 nm resolution typical for SAXS. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. An analysis of the experimental and published data made it possible to hypothesize that just before the Dps expression, it is IHF that forms toroidal structures previously observed in vivo and prepares the platform for formation of DNA–Dps crystals. The results obtained are necessary for further investigation of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome resistance of various pathogens to external conditions.

Nucleoid-associated proteins (NAPs) control the structure and functions of bacterial nucleoid. Histone-like HU proteins are most abundant NAPs in dividing bacterial cells. Previously, structural ensembles of conformations of HU proteins from pathogenic mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum were obtained using NMR spectroscopy. A structural study of these mycoplasma proteins is performed by small-angle X-ray scattering (SAXS). The occurrence of individual conformations from the ensemble, obtained by NMR, is estimated from the scattering data on HU protein solutions. In particular, an approach based on characterization of equilibrium mixtures in terms of volume fractions of their components was applied. The general shape of the proteins and their oligomeric state are independently confirmed using ab initio bead modelling. The flexibility of DNA-binding protein domains is analyzed by the ensemble optimization method, which is based on comparison of the structural characteristics of conformations fitting the SAXS data to the distribution of these characteristics in a randomly generated set. The results obtained give a new insight on the variability of the structure of HU proteins, which is necessary for their functioning.

DNA-binding protein from starved cells (Dps) takes a special place among dodecamer mini-ferritins. Its most important function is protection of bacterial genome from various types of destructive external factors via in cellulo Dps-DNA co-crystallization. This protective response results in the emergence of bacterial resistance to antibiotics and other drugs. The protective properties of Dps have attracted a significant attention of researchers. However, Dps has another equally important functional role. Being a ferritin-like protein, Dps acts as an iron depot and protects bacterial cells from the oxidative damage initiated by the excess of iron. Here we investigated formation of iron oxide nanoparticles in the internal cavity of the Dps dodecamer. We used anomalous small-angle X-ray scattering as the main research technique, which allows to examine the structure of metal-containing biological macromolecules and to analyze the size distribution of metal nanoparticles formed in them. The contributions of protein and metal components to total scattering were distinguished by varying the energy of the incident X-ray radiation near the edge of the metal atom absorption band (the K-band for iron). We examined Dps specimens containing 50, 500, and 2000 iron atoms per protein dodecamer. Analysis of the particle size distribution showed that, depending on the iron content in the solution, the size of the nanoparticles formed inside the protein molecule was 2 to 4 nm and the growth of metal nanoparticles was limited by the size of the protein inner cavity. We also found some amount of iron ions in the Dps surface layer. This layer is very important for the protein to perform its protective functions, since the surface-located N-terminal domains determine the nature of interactions between Dps and DNA. In general, the results obtained in this work can be useful for the next step in studying the Dps phenomenon, as well as in creating biocompatible and solution-stabilized metal nanoparticles.

The Ensemble Optimization Method (EOM) is a popular approach to describe small-angle X-ray scattering (SAXS) data from highly disordered proteins. The EOM algorithm selects subensembles of coexisting states from large pools of randomized conformations to fit the SAXS data. Based on the unphysical bimodal radius of gyration (Rg) distribution of conformations resulting from the EOM analysis, a recent article (Fagerberg et al. J. Chem. Theory Comput. 2019, 15 (12), 6968-6983) concluded that this approach inadequately described the SAXS data measured for human Histatin 5 (Hst5), a peptide with antifungal properties. Using extensive experimental and synthetic data, we explored the origin of this observation. We found that the one-bead-per-residue coarse-grained representation with averaged scattering form factors (provided in the EOM as an add-on to represent disordered missing loops or domains) may not be appropriate for EOM analyses of scattering data from short (below 50 residues) proteins/peptides. The method of choice for these proteins is to employ atomistic models (e.g., from molecular dynamics simulations) to sample the protein conformational landscape. As a convenient alternative, we have also improved the coarse-grained approach by introducing amino acid specific form factors in the calculations. We also found that, for small proteins, the search for relatively large subensembles of 20-50 conformers (as implemented in the original EOM version) more adequately describes the conformational space sampled in solution than the procedures optimizing the ensemble size. Our observations have been added as recommendations into the information for EOM users to promote the proper utilization of the program for ensemble-based modeling of SAXS data for all types of disordered systems.

Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.IMPORTANCE The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to the Avulavirus subfamily of the Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery.

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen.

A series of carbosilane dendrimers of the 4th, 6th, and 7th generations with a terminal trimethylsilylsiloxane layer was synthesized. Theoretical models of these dendrimers were developed, and equilibrium dendrimer conformations obtained via molecular dynamics simulations were in a good agreement with experimental small-angle X-ray scattering (SAXS) data demonstrating molecule monodispersity and an almost spherical shape. It was confirmed that the glass transition temperature is independent of the dendrimer generation, but is greatly affected by the chemical nature of the dendrimer terminal groups. A sharp increase in the zero-shear viscosity of dendrimer melts was found between the 5th and the 7th dendrimer generations, which was qualitatively identical to that previously reported for polycarbosilane dendrimers with butyl terminal groups. The viscoelastic properties of high-generation dendrimers seem to follow some general trends with an increase in the generation number, which are determined by the regular branching structure of dendrimers.

The ATSAS software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the ATSAS 3.0 package are described. They include IMSIM , for simulating isotropic 2D scattering patterns; IMOP , to perform operations on 2D images and masks; DATRESAMPLE , a method for variance estimation of structural invariants through parametric resampling; DATFT , which computes the pair distance distribution function by a direct Fourier transform of the scattering data; PDDFFIT , to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in DATMW for Bayesian consensus-based concentration-independent molecular weight estimation; DATMIF , an ab initio shape analysis method that optimizes the search model directly against the scattering data; DAMEMB , an application to set up the initial search volume for multiphase modelling of membrane proteins; ELLLIP , to perform quasi-atomistic modelling of liposomes with elliptical shapes; NMATOR , which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; DAMMIX , which reconstructs the shape of an unknown intermediate in an evolving system; and LIPMIX and BILMIX , for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the PRIMUS graphical interface to Qt5, updating SASpy – a PyMOL plugin to run a subset of ATSAS tools – to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future ATSAS releases. All these features are implemented in ATSAS 3.0 , freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.

The construction of three-dimensional models of protein macromolecules is a serious challenge due to the possible ambiguity of solving the inverse problem of reconstructing a three-dimensional structure from a one-dimensional small-angle scattering profile. The target function of this task can have several local minima, which leads to the dependence of the solution on the initial values of the model parameters and on the method of finding the global minimum. The problem of creating structural models is also complicated by averaging the scattering pattern over all orientations of particles in space and by the size and shape distribution of scattering objects in the case of polydispersity and/or polymorphism. In this study, the issue of ambiguity in solving inverse problems and restoring the three-dimensional structure of a protein is considered using the structure of the ectodomain of an insulin receptor-related receptor (ectoIRR) in solution as an example. The paper presents a consistent approach to solving this problem, starting from the determination of general structural parameters and ab initio reconstruction of shape to modeling by rigid bodies (using molecular tectonics), hybrid methods, and analysis of scattering profiles by singular vector decomposition.

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA-Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA-Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA-Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA-Dps complexes. Angle analysis has demonstrated that in DNA-Dps complexes a Dps molecule contacts with a DNA segment of similar to 6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet. (C) 2021 Elsevier Ltd. All rights reserved.