Signal to noise considerations for single crystal femtosecond time resolved crystallography of the Photoactive Yellow Protein

Abstract

Femtosecond time resolved pump-probe protein X-ray crystallography requires highly accurate measurements of the photoinduced structure factor amplitude differences. In the case of femtosecond photolysis of single P63 crystals of the Photoactive Yellow Protein, it is shown that photochemical dynamics place a considerable restraint on the achievable time resolution due to the requirement to stretch and add second order dispersion in order to generate threshold concentration levels in the interaction region. Here, we report on using a 'quasi-cw' approach to use the rotation method with monochromatic radiation and 2 eV bandwidth at 9.465 keV at the Linac Coherent Light Source operated in SASE mode. A source of significant Bragg reflection intensity noise is identified from the combination of mode structure and jitter with very small mosaic spread of the crystals and very low convergence of the XFEL source. The accuracy with which the three dimensional reflection is approximated by the 'quasi-cw' rotation method with the pulsed source is modelled from the experimentally collected X-ray pulse intensities together with the measured rocking curves. This model is extended to predict merging statistics for recently demonstrated self seeded mode generated pulse train with improved stability, in addition to extrapolating to single crystal experiments with increased mosaic spread. The results show that the noise level can be adequately modelled in this manner, indicating that the large intensity fluctuations dominate the merged signal-to-noise (I/σI) value. Furthermore, these results predict that using the self seeded mode together with more mosaic crystals, sufficient accuracy may be obtained in order to resolve typical photoinduced structure factor amplitude differences, as taken from representative synchrotron results.

Watching proteins function with time-resolved x-ray crystallography

“…For macroscopic crystals a step-scan technique as employed in monochromatic neutron scattering (Niimura et al ., 1997) may be adapted (Suga et al ., 2015). Due to the inherent intensity and energy fluctuations of the XFEL beam and damaging ultra-high X-ray intensity, a time-resolved pump-probe experiment with macroscopic crystals is a challenge (van Thor et al ., 2014). Using nano- and micro-crystals with a serial crystallography approach proved to be the most suitable method for data collection at XFELs, both for static and time-resolved measurements (use of nano-crystals is an exception; most XFEL experiments used micro-crystals).…”

Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

“…S2) corresponding to higher experimental noise in the structure amplitudes. If the noise in the amplitudes is too high, DED features deteriorate (29, 30). To extract faint DED features associated with small structural changes of PYP, diffraction patterns should preferably be collected with a laser-on, laser-off sequence to accumulate an equal number of light and dark patterns, aiming for redundancies on the order of 1500 in the highest resolution shell.…”

Applications and Limits of Time-to-Energy Mapping of Protein Crystal Diffraction Using Energy-Chirped Polychromatic XFEL Pulses

“…As this is considerably smaller than the mosaic spread of protein crystals, the latter is the determining factor. The range of mosaic spread used here considers very low mosaicity values, such as those from crystals of the photoactive yellow protein (PYP, η ≈ 0.04 • [56]), as well as values up to ten times larger. From the BW req of a reflection, it is also possible to calculate the amount of time it is stimulated for (Figure 4b).…”