X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature.
File(s)ijms-18-01918-v3.pdf (3.39 MB)
Published version
Author(s)
Type
Journal Article
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.
Date Issued
2017-09-07
Date Acceptance
2017-09-02
Citation
International Journal of Molecular Sciences, 2017, 18 (9)
ISSN
1422-0067
Publisher
MDPI AG
Journal / Book Title
International Journal of Molecular Sciences
Volume
18
Issue
9
Copyright Statement
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).
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/28880248
PII: ijms18091918
Subjects
LCLS
SACLA
SFX
Skylan-NS
XFEL
rsFP
Publication Status
Published online
Coverage Spatial
Switzerland
Article Number
1918