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

Journal Article · · International Journal of Molecular Sciences (Online)
DOI:https://doi.org/10.3390/ijms18091918· OSTI ID:1628376
 [1];  [1];  [1];  [2];  [1];  [1];  [1];  [1];  [3];  [1];  [1];  [4];  [4];  [4];  [4];  [5];  [5];  [6];  [7];  [1]
  1. Imperial College, London (United Kingdom)
  2. Univ. of Tokyo (Japan)
  3. Science and Technology Facilities Council (STFC), Oxford (United Kingdom). Diamond Light Source, Ltd.
  4. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
  5. RIKEN SPring-8 Center, Hyogo (Japan)
  6. RIKEN SPring-8 Center, Hyogo (Japan); Japan Synchrotron Radiation Research Institute, Sayo, Hyogo (Japan)
  7. RIKEN SPring-8 Center, Hyogo (Japan); Kyoto Univ. (Japan)
+ Show Author Affiliations

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.

Research Organization:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
AC02-76SF00515
OSTI ID:
1628376
Journal Information:
International Journal of Molecular Sciences (Online), Vol. 18, Issue 9; ISSN 1422-0067
Publisher:
MDPICopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 12 works
Citation information provided by
Web of Science

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Cited By (7)

Strategies for sample delivery for femtosecond crystallography text January 2019
Climbing the Data Mountain: Processing of SFX Data text January 2018
A guide to sample delivery systems for serial crystallography journal August 2019
Climbing the Data Mountain: Processing of SFX Data book January 2018
Viscosity-adjustable grease matrices for serial nanocrystallography journal January 2020
Optical control, selection and analysis of population dynamics in ultrafast protein X-ray crystallography
  • Hutchison, Christopher D. M.; van Thor, Jasper J.
  • Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, Vol. 377, Issue 2145 https://doi.org/10.1098/rsta.2017.0474
journal April 2019
Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography journal May 2018

Figures / Tables (9)

Table 1(p. 3)table Table 1
Figure 1(p. 4)figure Figure 1
Figure 2(p. 5)figure Figure 2
Figure 3(p. 6)figure Figure 3
Figure 4(p. 7)figure Figure 4
Table 2(p. 10)table Table 2
Figure A1(p. 11)figure Figure A1
Figure A2(p. 12)figure Figure A2
Figure A3(p. 12)figure Figure A3

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