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

Author:

van Thor Jasper J.1234,Warren Mark M.1234,Lincoln Craig N.1234,Chollet Matthieu567,Lemke Henrik Till567,Fritz David M.567,Schmidt Marius8910,Tenboer Jason8910,Ren Zhong111213,Srajer Vukica111213,Moffat Keith1415161112,Graber Tim111213

Affiliation:

1. Imperial College London

2. Division of Molecular Biosciences

3. South Kensington Campus

4. London SW7 2AZ, UK

5. LCLS

6. SLAC National Accelerator Laboratory

7. Menlo Park, USA

8. Department of Physics

9. University of Wisconsin-Milwaukee

10. Milwaukee, USA

11. Center for Advanced Radiation Sources

12. The University of Chicago

13. Chicago, USA

14. Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics

15. University of Chicago

16. Chicago, United States

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 P63crystals 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.

Publisher

Royal Society of Chemistry (RSC)

Subject

Physical and Theoretical Chemistry

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