Appraising protein conformational changes by resampling time-resolved serial x-ray crystallography data.

With the development of serial crystallography at both x-ray free electron laser and synchrotron radiation sources, time-resolved x-ray crystallography is increasingly being applied to study conformational changes in macromolecules. A successful time-resolved serial crystallography study requires the growth of microcrystals, a mechanism for synchronized and homogeneous excitation of the reaction of interest within microcrystals, and tools for structural interpretation. Here, we utilize time-resolved serial femtosecond crystallography data collected from microcrystals of bacteriorhodopsin to compare results from partial occupancy structural refinement and refinement against extrapolated data. We illustrate the domain wherein the amplitude of refined conformational changes is inversely proportional to the activated state occupancy. We illustrate how resampling strategies allow coordinate uncertainty to be estimated and demonstrate that these two approaches to structural refinement agree within coordinate errors. We illustrate how singular value decomposition of a set of difference Fourier electron density maps calculated from resampled data can minimize phase bias in these maps, and we quantify residual densities for transient water molecules by analyzing difference Fourier and Polder omit maps from resampled data. We suggest that these tools may assist others in judging the confidence with which observed electron density differences may be interpreted as functionally important conformational changes.