AlphaFold‐predicted protein structures and small‐angle X‐ray scattering: insights from an extended examination of selected data in the Small‐Angle Scattering Biological Data Bank

By providing predicted protein structures from nearly all known protein sequences, the artificial intelligence program AlphaFold (AF) is having a major impact on structural biology. While a stunning accuracy has been achieved for many folding units, predicted unstructured regions and the arrangement of potentially flexible linkers connecting structured domains present challenges. Focusing on single‐chain structures without prosthetic groups, an earlier comparison of features derived from small‐angle X‐ray scattering (SAXS) data taken from the Small‐Angle Scattering Biological Data Bank (SASBDB) is extended to those calculated using the corresponding AF‐predicted structures. Selected SASBDB entries were carefully examined to ensure that they represented data from monodisperse protein solutions and had sufficient statistical precision and q resolution for reliable structural evaluation. Three examples were identified where there is clear evidence that the single AF‐predicted structure cannot account for the experimental SAXS data. Instead, excellent agreement is found with ensemble models generated by allowing for flexible linkers between high‐confidence predicted structured domains. A pool of representative structures was generated using a Monte Carlo method that adjusts backbone dihedral allowed angles along potentially flexible regions. A fast ensemble modelling method was employed that optimizes the fit of pair distance distribution functions [P(r) versus r] and intensity profiles [I(q) versus q] computed from the pool to their experimental counterparts. These results highlight the complementarity between AF prediction, solution SAXS and molecular dynamics/conformational sampling for structural modelling of proteins having both structured and flexible regions. A rapid ensemble modelling method that optimizes the fit to the small‐angle X‐ray scattering (SAXS)‐derived pair‐wise distance distribution function [P(r) versus r] and the measured intensity profile [I(q) versus q] has been used to account for differences between AlphaFold‐predicted and experimental SAXS profiles. By considering the confidence levels that come with the predicted structures, a conformational ensemble with potentially flexible linkers between stable folded domains can be optimized to provide representative structures.