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Fixed-target pump-probe SFX: eliminating the scourge of light contamination.
Gotthard, Guillaume; Flores-Ibarra, Andrea; Carrillo, Melissa; Kepa, Michal W; Mason, Thomas J; Stegmann, Dennis P; Olasz, Bence; Pachota, Magdalena; Dworkowski, Florian; Ozerov, Dmitry; Pedrini, Bill F; Padeste, Celestino; Beale, John H; Nogly, Przemyslaw.
Afiliación
  • Gotthard G; Laboratory of Biomolecular Research, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Flores-Ibarra A; Dioscuri Center for Structural Dynamics of Receptors, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-380 Krakow, Poland.
  • Carrillo M; Laboratory of Nanoscale Biology, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Kepa MW; Laboratory of Biomolecular Research, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Mason TJ; Laboratory of Biomolecular Research, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Stegmann DP; Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Olasz B; Dioscuri Center for Structural Dynamics of Receptors, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-380 Krakow, Poland.
  • Pachota M; Dioscuri Center for Structural Dynamics of Receptors, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-380 Krakow, Poland.
  • Dworkowski F; Laboratory for Synchrotron Radiation and Femtochemistry, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Ozerov D; Science IT Infrastructure and Services, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Pedrini BF; Laboratory for X-ray Nanoscience and Technologies, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Padeste C; Laboratory of Nanoscale Biology, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Beale JH; Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, 5232 Villigen, Switzerland.
  • Nogly P; Institute of Molecular Biology and Biophysics, ETH Zurich, Rämistrasse 101, 8092 Zürich, Switzerland.
IUCrJ ; 11(Pt 5): 749-761, 2024 Sep 01.
Article en En | MEDLINE | ID: mdl-38980142
ABSTRACT
X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.
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Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Chlamydomonas reinhardtii Idioma: En Revista: IUCrJ Año: 2024 Tipo del documento: Article País de afiliación: Suiza

Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Chlamydomonas reinhardtii Idioma: En Revista: IUCrJ Año: 2024 Tipo del documento: Article País de afiliación: Suiza