Direct comparison of the crystal and solution structure of xylanase from Trichoderma longibrachiatum

The small angle X-ray scattering (SAXS) data of xylanase XYNII (endo-1,4-beta-xylan xylanohydrolase EC 3.2.1.8) from Trichoderma longibrachiatum, an enzyme catalysing the reaction of accidental hydrolysis of beta-1,4-D-xylosidic linkages of xylan, were recorded for protein solution using synchrotron radiation. The experimental data were compared with those of theoretical scattering calculated on the basis of the known crystal structure. The radius of gyration measured by SAXS (RG = 1.7 nm) was about 3.5% larger and the maximum dimension in the distance distribution function about 5 % larger than the corresponding values calculated on the basis of the crystal structure.     

Direct comparison of the crystal and solution structure of glucose/xylose isomerase from Streptomyces rubiginosus

Glucose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5.) catalyses the isomerization reaction of glucose and xylose. The small angle X-ray scattering (SAXS) data of glucose/xylose isomerase from Streptomyces rubiginosus were recorded for protein solution using synchrotron radiation. The experimental data were compared with theoretical scattering calculated on the basis of the known crystal structure (PDB code: 1OAD). The radius of gyration measured by SAXS (R(G)=3.30 nm) was almost identical and the maximum dimension in the distance distribution function was by about 2.5 % lower than the corresponding values calculated on the basis of the crystal structure.     

A solution SAXS study of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet.

The crystal structure of a soluble form of Borrelia burgdorferi outer surface protein A (OspA) complexed with the Fab fragment of a monoclonal antibody has revealed an unusual structure that has a repetitive antiparallel beta topology with a nonglobular, single layer beta-sheet connecting the globular N- and C-terminal domains. Earlier NMR studies have shown that the local structure of OspA including the single layer beta-sheet is similar to the crystal structure. Here we report a small angle X-ray scattering (SAXS) study of the global conformation of OspA in solution. The radius of gyration (Rg) and the length distribution function (P(r)) of OspA measured by SAXS in solution are nearly identical to the calculated ones from the crystal structure, respectively. The NMR and SAXS experiments complement each other to show that OspA including the central single-layer beta-sheet is a stable structure in solution, and that the OspA crystal structure represents the predominant solution conformation of the protein.     

Determination of protein oligomeric structure from small‐angle X‐ray scattering

Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.     

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.     

A systematic comparison of three structure determination methods from NMR data: Dependence upon quality and quantity of data

Summary We have systematically examined how the quality of NMR protein structures depends on (1) the number of NOE distance constraints. (2) their assumed precision, (3) the method of structure calculation and (4) the size of the protein. The test sets of distance constraints have been derived from the crystal structures of crambin (5 kDa) and staphylococcal nuclease (17 kDa). Three methods of structure calculation have been compared: Distance Geometry (DGEOM), Restrained Molecular Dynamics (XPLOR) and the Double Iterated Kalman Filter (DIKF). All three methods can reproduce the general features of the starting structure under all conditions tested. In many instances the apparent precision of the calculated structure (as measured by the RMS dispersion from the average) is greater than its accuracy (as measured by the RMS deviation of the average structure from the starting crystal structure). The global RMS deviations from the reference structures decrease exponentially as the number of constraints is increased, and after using about 30% of all potential constraints, the crrors asymptotically approach a limiting value. Increasing the assumed precision of the constraints has the same qualitative effect as increasing the number of constraints. For comparable numbers of constraints/residue, the precision of the calculated structure is less for the larger than for the smaller protein, regardless of the method of calculation. The accuracy of the average structure calculated by Restrained Molecular Dynamics is greater than that of structures obtained by purely geometric methods (DGEOM and DIKF).     

Solution structure of C-terminal Escherichia coli translation initiation factor IF2 by small-angle X-ray scattering

Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule. Furthermore, the SAXS data revealed that E. coli IF2 in solution adopts a structure that is significantly different from the crystal structure of orthologous aIF5B from Methanobacterium thermoautotrophicum. This crystal structure constitutes the only atomic resolution structural knowledge of the full-length factor. Computer programs were applied to the SAXS data to provide an initial structural model for IF2 in solution. The low-resolution nature of SAXS prevents the elucidation of a complete and detailed structure, but the resulting model for C-terminal E. coli IF2 indicates important structural differences between the aIF5B crystal structure and IF2 in solution. The chalice-like structure with a highly exposed alpha-helical stretch observed for the aIF5B crystal structure was not found in the structural model of IF2 in solution, in which domain VI-2 is moved closer to the rest of the protein.     

Structural insights into the beta-mannosidase from T. reesei obtained by synchrotron small-angle X-ray solution scattering enhanced by X-ray crystallography

A molecular envelope of the beta-mannosidase from Trichoderma reesei has been obtained by combined use of solution small-angle X-ray scattering (SAXS) and protein crystallography. Crystallographic data at 4 A resolution have been used to enhance informational content of the SAXS data and to obtain an independent, more detailed protein shape. The phased molecular replacement technique using a low resolution SAXS model, building, and refinement of a free atom model has been employed successfully. The SAXS and crystallographic free atom models exhibit a similar globular form and were used to assess available crystallographic models of glycosyl hydrolases. The structure of the beta-galactosidase, a member of a family 2, clan GHA glycosyl hydrolases, shows an excellent fit to the experimental molecular envelope and distance distribution function of the beta-mannosidase, indicating gross similarities in their three-dimensional structures. The secondary structure of beta-mannosidase quantified by circular dichroism measurements is in a good agreement with that of beta-galactosidase. We show that a comparison of distance distribution functions in combination with 1D and 2D sequence alignment techniques was able to restrict the number of possible structurally homologous proteins. The method could be applied as a general method in structural genomics and related fields once protein solution scattering data are available.     

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.     

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     

Small-angle X-ray scattering reveals structural dynamics of the botulinum neurotoxin associating protein, nontoxic nonhemagglutinin

In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.     

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.     

CH2 domain orientation of human immunoglobulin G in solution: Structural comparison of glycosylated and aglycosylated Fc regions using small-angle X-ray scattering

The N-linked glycan in immunoglobulin G is critical for the stability and function of the crystallizable fragment (Fc) region. Alteration of these protein properties upon the removal of the N-linked glycan has often been explained by the alteration of the C_H2 domain orientation in the Fc region. To confirm this hypothesis, we examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution. Conformational characteristics of the C_H2 domain orientation were validated by comparison with SAXS profiles theoretically calculated from multiple crystal structures of the Fc region with different C_H2 domain orientations. The reduced chi-square values from the fitting analyses of gFc and aFc associated with the degree of openness or closure of each crystal structure, as determined from the first principal component that partially governed the variation of the C_H2 domain orientation extracted by a singular value decomposition analysis. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a “semi-closed” C_H2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the C_H2 domain orientation in solution. These findings will guide the development of methodology for the production of highly refined functional Fc variants.     

The linker of calmodulin lacking Glu84 is elongated in solution,although it is bent in the crystal

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 Å and 62.5 Å, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 Å and 55.0 Å, and smaller than those expected from the crystal structure of wild type, 22.8 Å and 67.5 Å. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether. © 1996 Wiley-Liss, Inc.     

The subdomain structure of human serum albumin in solution under different pH conditions studied by small angle X-ray scattering

Small-angle X-ray scattering (SAXS) was used to study structural characteristics of human serum albumin (HSA) in solution under different pH conditions. Guinier analysis of SAXS results yielded values of the molecular radius of gyration ranging from 26.7 Å to 34.5 Å for pH varying from 2.5 to 7.0. This suggests the existence of significant differences in the overall shape of the molecule at different pH. Molecular models based on subdomains with different spatial configurations were proposed. The distance distribution functions associated with these models were calculated and compared with those determined from the experimental SAXS intensity functions. The conclusion of this SAXS study is that the arrangement of molecular subdomains is clearly pH dependent; the molecule adopting more or less compact configuration for different pH conditions. The conclusions of this systematic study on the modification in molecular shape of HSA as a response to pH changes is consistent with those of previous investigations performed for particular pH conditions. Correspondence to: J. R. Olivieri     

The allosteric effector l-lactate induces a conformational change of 2x6-meric lobster hemocyanin in the oxy state as revealed by small angle x-ray scattering

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.     

Structure study of cellulose fibers wet-spun from environmentally friendly NaOH/urea aqueous solutions

In this study, structure changes of regenerated cellulose fibers wet-spun from a cotton linter pulp (degree of polymerization approximately 620) solution in an NaOH/urea solvent under different conditions were investigated by simultaneous synchrotron wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS). WAXD results indicated that the increase in flow rate during spinning produced a better crystal orientation and a higher degree of crystallinity, whereas a 2-fold increase in draw ratio only affected the crystal orientation. When coagulated in a H2SO4/Na2SO4 aqueous solution at 15 degrees C, the regenerated fibers exhibited the highest crystallinity and a crystal orientation comparable to that of commercial rayon fibers by the viscose method. SAXS patterns exhibited a pair of meridional maxima in all regenerated cellulose fibers, indicating the existence of a lamellar structure. A fibrillar superstructure was observed only at higher flow rates (>20 m/min). The conformation of cellulose molecules in NaOH/urea aqueous solution was also investigated by static and dynamic light scattering. It was found that cellulose chains formed aggregates with a radius of gyration, Rg, of about 232 nm and an apparent hydrodynamic radius, Rh, of about 172 nm. The NaOH/urea solvent system is low-cost and environmentally friendly, which may offer an alternative route to replace more hazardous existing methods for the production of regenerated cellulose fibers.     

Small angle X-ray scattering-assisted protein structure prediction in CASP13 and emergence of solution structure differences

Small angle X-ray scattering (SAXS) measures comprehensive distance information on a protein's structure, which can constrain and guide computational structure prediction algorithms. Here, we evaluate structure predictions of 11 monomeric and oligomeric proteins for which SAXS data were collected and provided to predictors in the 13th round of the Critical Assessment of protein Structure Prediction (CASP13). The category for SAXS-assisted predictions made gains in certain areas for CASP13 compared to CASP12. Improvements included higher quality data with size exclusion chromatography-SAXS (SEC-SAXS) and better selection of targets and communication of results by CASP organizers. In several cases, we can track improvements in model accuracy with use of SAXS data. For hard multimeric targets where regular folding algorithms were unsuccessful, SAXS data helped predictors to build models better resembling the global shape of the target. For most models, however, no significant improvement in model accuracy at the domain level was registered from use of SAXS data, when rigorously comparing SAXS-assisted models to the best regular server predictions. To promote future progress in this category, we identify successes, challenges, and opportunities for improved strategies in prediction, assessment, and communication of SAXS data to predictors. An important observation is that, for many targets, SAXS data were inconsistent with crystal structures, suggesting that these proteins adopt different conformation(s) in solution. This CASP13 result, if representative of PDB structures and future CASP targets, may have substantive implications for the structure training databases used for machine learning, CASP, and use of prediction models for biology.