Conformational ensembles of flexible multidomain proteins: How close are we to accurate and reliable predictions?

Multidomain proteins connected by flexible linkers populate conformational ensembles that are challenging to characterize using conventional structural biology methods. In domain-linker-domain (DLD) proteins, linker-mediated inter-domain relative positions and orientations are functionally relevant, yet their dynamical behavior in solution normally remain poorly described. Small-angle X-ray scattering (SAXS) provides ensemble-averaged structural information for such systems; however, coupling with computational modeling is required to accurately describe the dynamic behavior of this family of proteins in solution. Here, we present a systematic evaluation of five ensemble-generation strategies applied to a set of eighteen proteins sharing the same two globular domains, connected by naturally occurring linkers of varying length and composition. Modeling methods based on different underlying principles are compared by assessing their agreement to experimental SAXS data, showing a large disparity and systematic structural biases among them. Furthermore, for each approach, we examine the effect of refinement against SAXS restraints and assess its capacity to describe the experimental data, as well as the induced biases in global dimensions and inter-domain distance distributions. This analysis underlines the importance of the initial conformational pool for deriving experimentally compatible ensembles. Overall, this work provides a high-quality benchmark for SAXS-driven ensemble modeling of flexible, multidomain proteins and establishes a framework for the critical interpretation of solution scattering data in systems with pronounced conformational heterogeneity.