Crystal optics select weak response of ultrafast crystallography of the Photoactive Yellow Protein

Ultrafast crystallography of light-induced processes can directly capture structural differences driven by short-pulse laser illumination. With short pump-probe observation time and short laser pulse durations, the electronic and nuclear dephasing times determine the extent and amplitude of third-order non-linear response in the measurements. While the third-order molecular response is unavoidable in ultrafast crystallography it is currently unknown to what extent non-linear optical excitation processes could contribute to the data. We present a separation of weak and strong response from pump-probe data of photoactive yellow protein crystals collected at a single laser power, based on a method of indexing transformation followed by optical crystallography. We show that this allows precise separation of the heterogeneous photochemical populations with a very high level of data correlation that is intrinsic from the methodology of serial crystallography data collection. This data correlation supports measurable crystallographic electron density differences for the cis-chromophore photoproduct at 3 ps delay comparing weak and strong interactions. While the resulting coordinate refinements are supported by these differences, their sub-Angstrom modifications are inconsequential when the RMS differences of displacements are considered at room temperature. We conclude that these minor crystallographic differences reflect the higher population and therefore signal-to-noise in the strong interaction dataset. Moreover, radical formation driven by excited state absorption, which is the dominating non-linear photoexcitation process under the conditions used, are not detected in high-quality and high-resolution TR-SFX of the photoactive yellow protein.