Fixed-target pump–probe SFX: eliminating the scourge of light contamination

Gotthard, G.; Flores-Ibarra, A.; Carrillo, M.; Kepa, M.W.; Mason, T.J.; Stegmann, D.P.; Olasz, B.; Pachota, M.; Dworkowski, F.; Ozerov, D.; Pedrini, B.F.; Padeste, C.; Beale, J.H.; Nogly, P.

doi:10.1107/S2052252524005591

Figure 4
Chip placement for laser incidence and Fourier difference electron-density maps for TR-SFX with CrLOV1 at Δt = 10 µs. Three different setups were investigated. Shown in the upper row is a transparent chip with cavity facing the pump pulse. This geometry yields an activation signal in all three maps, corresponding to thioether bond formation between the active-site cysteine and FMN: (a) F_obs^{interleaved-dark} − F_obs^laser-off, (b) F_obs^{interleaved-light} − F_obs^laser-off and (c) F_obs^{interleaved-light} − F_obs^{interleaved-dark}. The middle row shows that data collection using the opaque chip with cavity facing the pump pulse yields an activation signal in all three maps: (d) F_obs^{interleaved-dark} − F_obs^laser-off, (e) F_obs^{interleaved-light} − F_obs^laser-off and (f) F_obs^{interleaved-light} − F_obs^{interleaved-dark}. The bottom row shows that data acquisition using the opaque chip with aperture facing the pump pulse yields an activation signal where expected, while the lack of signal in the F_obs^{interleaved-dark} − F_obs^laser-off map indicates that light contamination was avoided in this setup: (g) F_obs^{interleaved-dark} − F_obs^laser-off, (h) F_obs^{interleaved-light} − F_obs^laser-off and (i) F_obs^{interleaved-light} − F_obs^{interleaved-dark}. The Fourier difference electron-density maps show positive and negative densities as blue and gold mesh, respectively, highlighting differences between datasets. The cartoon and sticks representation shows in light grey the dark state of the protein with its FMN ligand adjacent to but not covalently bound to Cys57. As a reference, the photoactivated late photocycle intermediate with FMN covalently bound to Cys57 is shown in purple. A schematic representation of the chip (grey) with a crystal sample (pink/blue) placement with respect to pump laser (blue) and XFEL pulses (red) is shown at the very left. The bottom panel shows the practical arrangement of the data from the wells contributing to the different maps.

IUCrJ

Volume 11| Part 5| September 2024|

ISSN: 2052-2525

https://doi.org/10.1107/S2052252524005591

PHYSICS | FELS

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