Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments

Abstract

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.

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

“…B, comparison of the calculated scattering curve of the model (red line) with the experimental scattering data (black line). Theoretical scattering curve was calculated and fitted to experimental data using FoXS (47). C, ab initio molecular envelope of the pASK1-CD⅐14-3-3 complex calculated from SAXS data (represented as a gray envelope) with superimposed single-state model of the complex.…”

“…Calculated molecular envelopes were aligned to structural models using the program SUPCOMB (45). Theoretical scattering curves were calculated from structural models and fitted to experimental scattering data using the programs CRYSOL (46) and FoXS (47).…”

“…Simulations consisting of 10 -30 runs (generating 101 conformations for each run) were allowed to sample the most probable conformations consistent with the starting structures and used distances derived from intermolecular cross-links (Table 3, cross-links #1-4) restrained to 20 Ϯ 8 Å from C␣ to C␣. Multistate modeling of the pASK1-CD⅐14-3-3 complex was performed using the MultiFoXS web server with the same starting structure (24,47). The ASK1-CD regions 659 -669 and 941-973 (with the exception of restrained 14-3-3 binding motif of one ASK1-CD protomer, region 963-968) were defined as flexible regions.…”

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

“…SAXS and SANS have the advantage that the respective contribution of the hydration layer (or shell) to the overall scattering from the macromolecules varies both in magnitude and sign (34). The HS contribution has been implemented in a number of programs to back-calculate SAXS (and, more rarely, SANS) curves from atomic structures, either as a homogeneous shell of a specific thickness (30)(31)(32)35), as grid elements (36,37), as dummy atoms (38,39), as explicit water molecules (40)(41)(42)(43)(44)(45), as a density map (46,47), or by voxelization (48). Accurate description of the HS is essential for the growing field of quasiatomic protein-structure modeling from solution data (34).…”

“…Accurate description of the HS is essential for the growing field of quasiatomic protein-structure modeling from solution data (34). Surprisingly, as more and more programs are being developed (38), new original experimental small-angle scattering (SAS) data describing HSs are rather scarce and are limited to a small number of protein systems. In particular, systematic studies using both SAXS and SANS to probe the HS as a function of physicochemical surface properties from a single protein system have, to our knowledge, not been carried out to date.…”