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Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL

Abstract

We report a method of femtosecond crystallography for solving radiation damage–free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-Å radiation damage–free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

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Figure 1: Schematic of the data collection approach using femtosecond crystallography at SACLA.
Figure 2: The O2-reduction site free of radiation damage.

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Acknowledgements

This work was supported by the X-ray Free Electron Laser Priority Strategy Program (The Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT)) (T.O., J.-R.S. and H.A.), the JST/CREST (K.H. and T.T.) and a grant-in-aid for Specially Promoted Research no. 24000018 from MEXT/Japan Society for the Promotion of Science (JSPS) (J.-R.S.). The XFEL experiments were performed at the beamline 3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos. 2012A8011, 2012B8040, 2013A8047 and 2013B8052). We thank G. Murshudov for his modification of REFMAC.

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Contributions

All authors contributed to the diffraction experiments at SACLA and discussed and commented on the results and the manuscript. K.H. and K.S.-I. planned experiments. K.T., Y.I., M. Yabashi and T.I. contributed to beamline operation. G.U., T.H., H.M. and M. Yamamoto contributed to development of the experimental instruments. N.Y., S.T., K.K., M.H., K.M., T.K. and E.Y. conducted protein purification, crystallization and diffraction experiments. S.Y. and T.T. wrote the manuscript. J.-R.S., T.O., H.S. and H.A. organized the damage-free diffraction experiment at SACLA.

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Correspondence to Shinya Yoshikawa or Hideo Ago.

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The authors declare no competing financial interests.

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Supplementary Figures 1 and 2, Supplementary Tables 1–5, Supplementary Results and Supplementary Note (PDF 924 kb)

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Hirata, K., Shinzawa-Itoh, K., Yano, N. et al. Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL. Nat Methods 11, 734–736 (2014). https://doi.org/10.1038/nmeth.2962

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