Uniqueness of models from small-angle scattering data: the impact of a hydration shell and complementary NMR restraints

Abstract

Small-angle scattering (SAS) has witnessed a breathtaking renaissance and expansion over the past 15 years regarding the determination of biomacromolecular structures in solution. While important issues such as sample quality, good experimental practice and guidelines for data analysis, interpretation, presentation, publication and deposition are increasingly being recognized, crucial topics such as the uniqueness, precision and accuracy of the structural models obtained by SAS are still only poorly understood and addressed. The present article provides an overview of recent developments in these fields with a focus on the influence of complementary NMR restraints and of a hydration shell on the uniqueness of biomacromolecular models. As a first topic, the impact of incorporating NMR orientational restraints in addition to SAS distance restraints is discussed using a quantitative visual representation that illustrates how the possible conformational space of a two-body system is reduced as a function of the available data. As a second topic, the impact of a hydration shell on modelling parameters of a two-body system is illustrated, in particular on its inter-body distance. Finally, practical recommendations are provided to take both effects into account and promising future perspectives of SAS approaches are discussed.

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

“…The R G values of all three GFPs in 100% D 2 O were smaller than their counterparts determined by SAXS or SANS in 8% D 2 O by 1-3 Å . This is consistent with a denser HS, in agreement with previous findings in other systems (30,34). The quality of the fits was very good and was similar among the three GFPs (Fig.…”

“…Finally, the HS densities did not vary a lot between the top 10 structures of each construct (Table 1) nor between the structures with poorer fits, which is a strong argument that the HS shell values are stable and independent from the specific terminus conformations. SANS data for the three hydrogenated GFP mutants were measured under different solvent contrast conditions of 8% D 2 O (coherent water SLD ¼ 0, i.e., equivalent to measuring proteins in vacuo (63)) and 100% D 2 O, where the influence of HS density on scattering curves is much more pronounced and of opposite contrast with respect to x-rays (34). Partially deuterated GFP(À6) and GFP(þ36) were also measured in 100% D 2 O (d-SANS), their theoretical match point (i.e., where GFP and solvent have the same average SLD), with the advantage of minimal incoherent solvent background.…”

“…It has been shown experimentally by SAXS/SANS that the solvent in the vicinity of proteins (within a few Å ngstroms) has an average SLD different from that of the bulk, usually slightly denser, that needs to be taken into account to accurately fit protein structures in aqueous solutions (19,30). 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).…”

“…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). 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.…”

Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

“…If one assumes a similar overall topology for the C-terminal parts of both systems the much higher relative decrease of R G observed here for PAN suggests a more pronounced relative displacement of the centres of mass of its six subunits upon nucleotide binding than the motions detected in ClpX. Furthermore, the parallel axis theorem3637 predicts that a rotation of a rigid subunit around its centre of mass does not change the overall radius of gyration of the complex it forms with other rigid partners. Therefore, a 10% reduction of the R G implies a significant translational displacement of subunit centres toward the centre of the PAN complex, possibly combined with a conformational contraction of individual subunits and involving motions of the N-terminal coiled-coil domains (Fig.…”