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Photoconvertible Fluorescent Proteins and the Role of Dynamics in Protein Evolution
Open AccessArticle

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

Molecular Biophysics, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
Protein Crystallography Facility, Centre for Structural Biology, Flowers Building, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Quantum Optics and Laser Science Group, Blackett Laboratory, Imperial College, London SW7 2AZ, UK
Diamond Light Source Ltd., Diamond House, Harwell Science & Innovation Campus, Didcot OX11 0DE, UK
LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Rd., Menlo Park, CA 94025, USA
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Hyogo 679-5148, Japan
Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Hyogo 679-5198, Japan
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2017, 18(9), 1918;
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)
PDF [3470 KB, uploaded 8 September 2017]


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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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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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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