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

DOI: 10.3390/ijms18091918 DOI Help

Authors: Christopher D. M. Hutchison (Imperial College London) , Violeta Cordon-preciado (Imperial College London) , Rhodri M. L. Morgan (Imperial College London) , Takanori Nakane (Imperial The University of TokyoLondon) , Josie Ferreira (Imperial College London) , Gabriel Dorlhiac (Imperial College London) , Alvaro Sanchez-gonzalez (Imperial College London) , Allan S. Johnson (Imperial College London) , Ann Fitzpatrick (Diamond Light Source) , Clyde Fare (Imperial College London) , Jon Marangos (Imperial College London) , Chun Hong Yoon (SLAC National Accelerator Laboratory) , Mark S. Hunter (Imperial College London) , Daniel P. Deponte (SLAC National Accelerator Laboratory) , S├ębastien Boutet (SLAC National Accelerator Laboratory) , Shigeki Owada (RIKEN SPring-8 Center) , Rie Tanaka (RIKEN SPring-8 Center) , Kensuke Tono (RIKEN SPring-8 Center; Japan Synchrotron Radiation Research Institute) , So Iwata (RIKEN SPring-8 Center; Kyoto University) , Jasper J. Van Thor (Imperial College London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: International Journal Of Molecular Sciences , VOL 18

State: Published (Approved)
Published: September 2017
Diamond Proposal Number(s): 12579

Open Access Open Access

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.

Journal Keywords: XFEL; SFX; rsFP; Skylan-NS; SACLA; LCLS

Subject Areas: Biology and Bio-materials


Instruments: B22-Multimode InfraRed imaging And Microspectroscopy , I03-Macromolecular Crystallography , I04-Macromolecular Crystallography