Single-Molecule X-ray Scattering Used to Visualize the Conformation Distribution of Biological Molecules via Single-Object Scattering Sampling

Biological macromolecules, the fundamental building blocks of life, exhibit dynamic structures in their natural environment. Traditional structure determination techniques often oversimplify these multifarious conformational spectra by capturing only ensemble- and time-averaged molecular structures. Addressing this gap, in this work, we extend the application of the single-object scattering sampling (SOSS) method to diverse biological molecules, including RNAs and proteins. Our approach, referred to as "Bio-SOSS", leverages ultrashort X-ray pulses to capture instantaneous structures. In Bio-SOSS, we employ two gold nanoparticles (AuNPs) as labels, which provide strong contrast in the X-ray scattering signal, to ensure precise distance determinations between labeled sites. We generated hypothetical Bio-SOSS images for RNAs, proteins, and an RNA-protein complex, each labeled with two AuNPs at specified positions. Subsequently, to validate the accuracy of Bio-SOSS, we extracted distances between these nanoparticle labels from the images and compared them with the actual values used to generate the Bio-SOSS images. Specifically, for a representative RNA (1KXK), the standard deviation in distance discrepancies between molecular dynamics snapshots and Bio-SOSS retrievals was found to be optimally around 0.2 A, typically within 1 A under practical experimental conditions at state-of-the-art X-ray free-electron laser facilities. Furthermore, we conducted an in-depth analysis of how various experimental factors, such as AuNP size, X-ray properties, and detector geometry, influence the accuracy of Bio-SOSS. This comprehensive investigation highlights the practicality and potential of Bio-SOSS in accurately capturing the diverse conformation spectrum of biological macromolecules, paving the way for deeper insights into their dynamic natures.