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Int. J. Mol. Sci. 2017, 18(9), 1918; https://doi.org/10.3390/ijms18091918

X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature

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Molecular Biophysics, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
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Protein Crystallography Facility, Centre for Structural Biology, Flowers Building, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
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Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
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Quantum Optics and Laser Science Group, Blackett Laboratory, Imperial College, London SW7 2AZ, UK
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Diamond Light Source Ltd., Diamond House, Harwell Science & Innovation Campus, Didcot OX11 0DE, UK
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LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Rd., Menlo Park, CA 94025, USA
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RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Hyogo 679-5148, Japan
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Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Hyogo 679-5198, Japan
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Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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Author to whom correspondence should be addressed.
Received: 14 August 2017 / Revised: 1 September 2017 / Accepted: 2 September 2017 / Published: 7 September 2017
(This article belongs to the Special Issue Fluorescent Proteins)
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Abstract

The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form. View Full-Text
Keywords: XFEL; SFX; rsFP; Skylan-NS; SACLA; LCLS XFEL; SFX; rsFP; Skylan-NS; SACLA; LCLS
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Hutchison, C.D.M.; Cordon-Preciado, V.; Morgan, R.M.L.; Nakane, T.; Ferreira, J.; Dorlhiac, G.; Sanchez-Gonzalez, A.; Johnson, A.S.; Fitzpatrick, A.; Fare, C.; Marangos, J.P.; Yoon, C.H.; Hunter, M.S.; DePonte, D.P.; Boutet, S.; Owada, S.; Tanaka, R.; Tono, K.; Iwata, S.; van Thor, J.J. X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature. Int. J. Mol. Sci. 2017, 18, 1918.

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