CASP10–BCL::Fold efficiently samples topologies of large proteins

During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template‐based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native‐like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE‐only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native‐like assembly of SSEs for further refinement and submission. It was also observed that for some β‐strand proteins model refinement failed as β‐strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non‐natural topologies that require loop regions to pass through the center of the protein. Proteins 2015; 83:547–563. © 2015 Wiley Periodicals, Inc.     

Solution structure of human and bovine beta(2)-glycoprotein I revealed by small-angle X-ray scattering

beta(2)-Glycoprotein I (beta(2)GPI) is a highly glycosylated phospholipid-binding plasma protein comprised of four complement control protein (CCP) domains and a distinct fifth domain. The structural organisation of human and bovine beta(2)GPI in aqueous solution was studied by small-angle X-ray scattering (SAXS). Low-resolution models that match the SAXS experimental data best were independently constructed by three different ab initio 3D-reconstruction algorithms. Similar elongated S-shaped models with distinct side-arms, which were correlated to the position of the carbohydrate chains, were restored from all three algorithms. Due to an additional glycosylation site located on the CCP2 domain of bovine beta(2)GPI a small change in the characteristic SAXS parameters was observed, which coincided with results obtained from SDS-PAGE. In comparison to the human analogue the corresponding restored low-resolution models displayed a similar S-shape with less bending in the middle part. As the experimental SAXS curves fit poorly to the simulated scattering curves calculated from the crystallographic coordinates of human beta(2)GPI, the crystal structure was modified. First, additional carbohydrate residues missing from the crystal structure were modelled. Second, on the basis of the low-resolution models, the J-shaped crystal structure was rotated between CCP3 and CCP2 assuming the greatest interdomain flexibility between these domains. An S-shaped model with a tilt angle of approximately 60 degrees between CCP3 and CCP2 yielded the best fit to the experimental SAXS data. Since there is evidence that beta(2)GPI can adopt different conformations, which reveal distinct differences in autoantibody recognition, our data clearly point to a reorientation of the flexible domains, which may be an essential feature for binding of autoantibodies.     

Reweighting of molecular simulations with explicit-solvent SAXS restraints elucidates ion-dependent RNA ensembles

Small-angle X-ray scattering (SAXS) experiments are increasingly used to probe RNA structure. A number of forward models that relate measured SAXS intensities and structural features, and that are suitable to model either explicit-solvent effects or solute dynamics, have been proposed in the past years. Here, we introduce an approach that integrates atomistic molecular dynamics simulations and SAXS experiments to reconstruct RNA structural ensembles while simultaneously accounting for both RNA conformational dynamics and explicit-solvent effects. Our protocol exploits SAXS pure-solute forward models and enhanced sampling methods to sample an heterogenous ensemble of structures, with no information towards the experiments provided on-the-fly. The generated structural ensemble is then reweighted through the maximum entropy principle so as to match reference SAXS experimental data at multiple ionic conditions. Importantly, accurate explicit-solvent forward models are used at this reweighting stage. We apply this framework to the GTPase-associated center, a relevant RNA molecule involved in protein translation, in order to elucidate its ion-dependent conformational ensembles. We show that (a) both solvent and dynamics are crucial to reproduce experimental SAXS data and (b) the resulting dynamical ensembles contain an ion-dependent fraction of extended structures.     

On the Calculation of SAXS Profiles of Folded and Intrinsically Disordered Proteins from Computer Simulations

Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms.     

Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments

A major challenge in structural biology is to characterize structures of proteins and their assemblies in solution. At low resolution, such a characterization may be achieved by small angle x-ray scattering (SAXS). Because SAXS analyses often require comparing profiles calculated from many atomic models against those determined by experiment, rapid and accurate profile computation from molecular structures is needed. We developed fast open-source x-ray scattering (FoXS) for profile computation. To match the experimental profile within the experimental noise, FoXS explicitly computes all interatomic distances and implicitly models the first hydration layer of the molecule. For assessing the accuracy of the modeled hydration layer, we performed contrast variation experiments for glucose isomerase and lysozyme, and found that FoXS can accurately represent density changes of this layer. The hydration layer model was also compared with a SAXS profile calculated for the explicit water molecules in the high-resolution structures of glucose isomerase and lysozyme. We tested FoXS on eleven protein, one DNA, and two RNA structures, revealing superior accuracy and speed versus CRYSOL, AquaSAXS, the Zernike polynomials-based method, and Fast-SAXS-pro. In addition, we demonstrated a significant correlation of the SAXS score with the accuracy of a structural model. Moreover, FoXS utility for analyzing heterogeneous samples was demonstrated for intrinsically flexible XLF-XRCC4 filaments and Ligase III-DNA complex. FoXS is extensively used as a standalone web server as a component of integrative structure determination by programs IMP, Chimera, and BILBOMD, as well as in other applications that require rapidly and accurately calculated SAXS profiles.     

Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments

A major challenge in structural biology is to characterize structures of proteins and their assemblies in solution. At low resolution, such a characterization may be achieved by small angle x-ray scattering (SAXS). Because SAXS analyses often require comparing profiles calculated from many atomic models against those determined by experiment, rapid and accurate profile computation from molecular structures is needed. We developed fast open-source x-ray scattering (FoXS) for profile computation. To match the experimental profile within the experimental noise, FoXS explicitly computes all interatomic distances and implicitly models the first hydration layer of the molecule. For assessing the accuracy of the modeled hydration layer, we performed contrast variation experiments for glucose isomerase and lysozyme, and found that FoXS can accurately represent density changes of this layer. The hydration layer model was also compared with a SAXS profile calculated for the explicit water molecules in the high-resolution structures of glucose isomerase and lysozyme. We tested FoXS on eleven protein, one DNA, and two RNA structures, revealing superior accuracy and speed versus CRYSOL, AquaSAXS, the Zernike polynomials-based method, and Fast-SAXS-pro. In addition, we demonstrated a significant correlation of the SAXS score with the accuracy of a structural model. Moreover, FoXS utility for analyzing heterogeneous samples was demonstrated for intrinsically flexible XLF-XRCC4 filaments and Ligase III-DNA complex. FoXS is extensively used as a standalone web server as a component of integrative structure determination by programs IMP, Chimera, and BILBOMD, as well as in other applications that require rapidly and accurately calculated SAXS profiles.     

Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.     

Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies

A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluster of many independently calculated solutions. To quantify how well the quaternary structures are determined based on their SAXS profiles, we used a benchmark of 12 simulated examples as well as an experimental SAXS profile of the homotetramer d-xylose isomerase. Optimization of the SAXS-dependent scoring function generally results in accurate models if sufficiently precise approximations for the constituent rigid bodies are available; otherwise, the best scoring models can have significant errors. Thus, SAXS profiles can play a useful role in the structural characterization of proteins and assemblies if they are combined with additional data and used judiciously. Our integration of a SAXS profile into modeling by satisfaction of spatial restraints will facilitate further integration of different kinds of data for structure determination of proteins and their assemblies.     

EFAMIX,a tool to decompose inline chromatography SAXS data from partially overlapping components

Small‐angle X‐ray scattering (SAXS) is an established technique for structural analysis of biological macromolecules in solution. During the last decade, inline chromatography setups coupling SAXS with size exclusion (SEC‐SAXS) or ion exchange (IEC‐SAXS) have become popular in the community. These setups allow one to separate individual components in the sample and to record SAXS data from isolated fractions, which is extremely important for subsequent data interpretation, analysis, and structural modeling. However, in case of partially overlapping elution peaks, inline chromatography SAXS may still yield scattering profiles from mixtures of components. The deconvolution of these scattering data into the individual fractions is nontrivial and potentially ambiguous. We describe a cross‐platform computer program, EFAMIX, for restoring the scattering and concentration profiles of the components based on the evolving factor analysis (EFA). The efficiency of the program is demonstrated in a number of simulated and experimental SEC‐SAXS data sets. Sensitivity and limitations of the method are explored, and its applicability to IEC‐SAXS data is discussed. EFAMIX requires minimal user intervention and is available to academic users through the program package ATSAS as from release 3.1.     

Characterization of proteins with wide-angle X-ray solution scattering (WAXS)

X-ray solution scattering in both the small-angle (SAXS) and wide-angle (WAXS) regimes is making an increasing impact on our understanding of biomolecular complexes. The accurate calculation of WAXS patterns from atomic coordinates has positioned the approach for rapid growth and integration with existing Structural Genomics efforts. WAXS data are sensitive to small structural changes in proteins; useful for calculation of the pair-distribution function at relatively high resolution; provides a means to characterize the breadth of the structural ensemble in solution; and can be used to identify proteins with similar folds. WAXS data are often used to test structural models, identify structural similarities and characterize structural changes. WAXS is highly complementary to crystallography and NMR. It holds great potential for the testing of structural models of proteins; identification of proteins that may exhibit novel folds; characterization of unfolded or natively disordered proteins; and detection of structural changes associated with protein function.     

Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.     

Lumbricus terrestris hemoglobin: A comparison of small-angle X-ray scattering and cryoelectron microscopy data

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of cylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm⁻¹ and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm⁻¹) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex. © John Wiley & Sons, Inc. Biopoly 45: 289–298, 1998     

Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.     

Structural and SAXS analysis of Tle5–Tli5 complex reveals a novel inhibition mechanism of H2‐T6SS in Pseudomonas aeruginosa

Widely spread in Gram‐negative bacteria, the type VI secretion system (T6SS) secretes many effector‐immunity protein pairs to help the bacteria compete against other prokaryotic rivals, and infect their eukaryotic hosts. Tle5 and Tle5B are two phospholipase effector protein secreted by T6SS of Pseudomonas aeruginosa. They can facilitate the bacterial internalization process into human epithelial cells by interacting with Akt protein of the PI3K‐Akt signal pathway. Tli5 and PA5086‐5088 are cognate immunity proteins of Tle5 and Tle5B, respectively. They can interact with their cognate effector proteins to suppress their virulence. Here, we report the crystal structure of Tli5 at 2.8Å resolution and successfully fit it into the Small angle X‐ray scattering (SAXS) model of the complete Tle5–Tli5 complex. We identified two important motifs in Tli5 through sequence and structural analysis. One is a conserved loop‐β‐hairpin motif that exists in the Tle5 immunity homologs, the other is a long and sharp α‐α motif that directly interacts with Tle5 according to SAXS data. We also distinguished the structural features of Tle5 and Tle5B family immunity proteins. Together, our work provided insights into a novel inhibition mechanism that may enhance our understanding of phospholipase D family proteins.     

A simple genetic algorithm for the optimization of multidomain protein homology models driven by NMR residual dipolar coupling and small angle <Emphasis Type="Italic">X</Emphasis>-ray scattering data

Most proteins comprise several domains and/or participate in functional complexes. Owing to ongoing structural genomic projects, it is likely that it will soon be possible to predict, with reasonable accuracy, the conserved regions of most structural domains. Under these circumstances, it will be important to have methods, based on simple-to-acquire experimental data, that allow to build and refine structures of multi-domain proteins or of protein complexes from homology models of the individual domains/proteins. It has been recently shown that small angle X-ray scattering (SAXS) and NMR residual dipolar coupling (RDC) data can be combined to determine the architecture of such objects when the X-ray structures of the domains are known and can be considered as rigid objects. We developed a simple genetic algorithm to achieve the same goal, but by using homology models of the domains considered as deformable objects. We applied it to two model systems, an S1KH bi-domain of the NusA protein and the γS-crystallin protein. Despite its simplicity our algorithm is able to generate good solutions when driven by SAXS and RDC data.     

Comparison of native and non‐native ubiquitin oligomers reveals analogous structures and reactivities

Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post‐translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non‐enzymatic methods to synthesize Ub chains of well‐defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non‐native linkages; however, few studies have examined the extent to which non‐native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non‐native linkages. Using small‐angle X‐ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non‐native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady‐state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non‐native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non‐native linkages can serve as surrogate substrates for explorations of Ub function.     

Macromolecular docking restrained by a small angle X-ray scattering profile

While many structures of single protein components are becoming available, structural characterization of their complexes remains challenging. Methods for modeling assembly structures from individual components frequently suffer from large errors, due to protein flexibility and inaccurate scoring functions. However, when additional information is available, it may be possible to reduce the errors and compute near-native complex structures. One such type of information is a small angle X-ray scattering (SAXS) profile that can be collected in a high-throughput fashion from a small amount of sample in solution. Here, we present an efficient method for protein–protein docking with a SAXS profile (FoXSDock): generation of complex models by rigid global docking with PatchDock, filtering of the models based on the SAXS profile, clustering of the models, and refining the interface by flexible docking with FireDock. FoXSDock is benchmarked on 124 protein complexes with simulated SAXS profiles, as well as on 6 complexes with experimentally determined SAXS profiles. When induced fit is less than 1.5 Å interface C_α RMSD and the fraction residues of missing from the component structures is less than 3%, FoXSDock can find a model close to the native structure within the top 10 predictions in 77% of the cases; in comparison, docking alone succeeds in only 34% of the cases. Thus, the integrative approach significantly improves on molecular docking alone. The improvement arises from an increased resolution of rigid docking sampling and more accurate scoring.     

Integrating NMR,SAXS, and Atomistic Simulations: Structure and Dynamics of a Two-Domain Protein

Multidomain proteins with two or more independently folded functional domains are prevalent in nature. Whereas most multidomain proteins are linked linearly in sequence, roughly one-tenth possess domain insertions where a guest domain is implanted into a loop of a host domain, such that the two domains are connected by a pair of interdomain linkers. Here, we characterized the influence of the interdomain linkers on the structure and dynamics of a domain-insertion protein in which the guest LysM domain is inserted into a central loop of the host CVNH domain. Expanding upon our previous crystallographic and NMR studies, we applied SAXS in combination with NMR paramagnetic relaxation enhancement to construct a structural model of the overall two-domain system. Although the two domains have no fixed relative orientation, certain orientations were found to be preferred over others. We also assessed the accuracies of molecular mechanics force fields in modeling the structure and dynamics of tethered multidomain proteins by integrating our experimental results with microsecond-scale atomistic molecular dynamics simulations. In particular, our evaluation of two different combinations of the latest force fields and water models revealed that both combinations accurately reproduce certain structural and dynamical properties, but are inaccurate for others. Overall, our study illustrates the value of integrating experimental NMR and SAXS studies with long timescale atomistic simulations for characterizing structural ensembles of flexibly linked multidomain systems.     

A Small Angle X-Ray Scattering Study of the Binding of Cyclosporin-A to Calmodulin

Small angle X-ray scattering (SAXS) was applied to the binding of the immunosuppressant drug cyclasporin-A to the protein calmodulin. Guinier analysis of the SAXS profiles yielded a radius of gyration, Rg, of 19.7 ± 0.3 Å for the native protein and 16.9 ± 0.3 Å for the drug/protein complex. Maximum entropy (maxent) methods of data analysis were used to calculate the distance distribution function, p(r). From this analysis, the R_g for the native protein is 20.9 ± 0.1 Å and that for the complex 16.7 ± 0.1 Å. The measured SAXS profiles and the derived p(r) for calmodulin agree with profiles calculated from the crystallographic structure of calmodulin. Major structural changes are induced in calmodulin on binding cyclosporin-A. A model consistent with the observed scattering profiles is an ellipsoid with major axes 55 and 36 Å. Molecular modeling of the calmodulin molecule suggests that bond rotation in the flexible α-helix linker region produces models consistent with the above observations.     

Validating Solution Ensembles from Molecular Dynamics Simulation by Wide-Angle X-ray Scattering Data

Wide-angle x-ray scattering (WAXS) experiments of biomolecules in solution have become increasingly popular because of technical advances in light sources and detectors. However, the structural interpretation of WAXS profiles is problematic, partly because accurate calculations of WAXS profiles from structural models have remained challenging. In this work, we present the calculation of WAXS profiles from explicit-solvent molecular dynamics (MD) simulations of five different proteins. Using only a single fitting parameter that accounts for experimental uncertainties because of the buffer subtraction and dark currents, we find excellent agreement to experimental profiles both at small and wide angles. Because explicit solvation eliminates free parameters associated with the solvation layer or the excluded solvent, which would require fitting to experimental data, we minimize the risk of overfitting. We further find that the influence from water models and protein force fields on calculated profiles are insignificant up to q ≈ 15 nm^?1. Using a series of simulations that allow increasing flexibility of the proteins, we show that incorporating thermal fluctuations into the calculations significantly improves agreement with experimental data, demonstrating the importance of protein dynamics in the interpretation of WAXS profiles. In addition, free MD simulations up to one microsecond suggest that the calculated profiles are highly sensitive with respect to minor conformational rearrangements of proteins, such as an increased flexibility of a loop or an increase of the radius of gyration by  <  1%. The present study suggests that quantitative comparison between MD simulations and experimental WAXS profiles emerges as an accurate tool to validate solution ensembles of biomolecules.