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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
oleh: Christopher D. M. Hutchison, Violeta Cordon-Preciado, Rhodri M. L. Morgan, Takanori Nakane, Josie Ferreira, Gabriel Dorlhiac, Alvaro Sanchez-Gonzalez, Allan S. Johnson, Ann Fitzpatrick, Clyde Fare, Jon P. Marangos, Chun Hong Yoon, Mark S. Hunter, Daniel P. DePonte, Sébastien Boutet, Shigeki Owada, Rie Tanaka, Kensuke Tono, So Iwata, Jasper J. van Thor
Format: | Article |
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Diterbitkan: | MDPI AG 2017-09-01 |
Deskripsi
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.