De novo structural ensemble determination from single-molecule X-ray scattering: A Bayesian approach

Abstract: Single molecule X-ray scattering experiments with free electron lasers have opened a new route to the structure determination of biomolecules. Because typically only very few photons per scattering image are recorded and thus the signal-to-noise ratio is very low in this extreme Poisson regime, structure refinement is quite challenging. In addition, in each scattering event the orientation of the biomolecule is random and unknown. As a further layer of complexity, many biomolecules show structural heterogeneity and conformational transitions between different distinct structures; these structural dynamics are averaged out by existing refinement methods. To overcome these limitations, here we developed and tested a rigorous Bayesian approach and demonstrate that it should be possible to determine not only a single structure, but an entire structural ensemble from these experiments. Using $10^6$ synthetic scattering images generated from molecular dynamics trajectories, our approach was able to resolve ensembles of eight alanine dipeptide conformers at $2\,$\r{A} resolution; similarly, we determined the unfolded ensemble of the protein chignolin at $4-7\,$\r{A} resolution using $1.2\cdot 10^7$ images. Unexpectedly, much fewer images are required to determine an ensemble of $n$ structures of $m$ atoms each than a single structure of $n\times m$ atoms, i.e., of the same total number of degrees of freedom. These findings show that X-ray scattering experiments using state-of-the-art free electron lasers should allow one to determine not only biomolecular structures, but whole structure ensembles and, ultimately, 'molecular movies'.