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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

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          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.

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          Linking crystallographic model and data quality.

          In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, R(merge) values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, R(merge) values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.
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            The fluorescent protein palette: tools for cellular imaging.

            This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems (227 references).
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              Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein.

              Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                07 September 2017
                September 2017
                : 18
                : 9
                : 1918
                Affiliations
                [1 ]Molecular Biophysics, Imperial College London, South Kensington Campus, London SW7 2AZ, UK; christopher.hutchison05@ 123456imperial.ac.uk (C.D.M.H.); violeta.cordon-preciado@ 123456imperial.ac.uk (V.C.-P.); j.ferreira14@ 123456imperial.ac.uk (J.F.); gabriel.dorlhiac13@ 123456imperial.ac.uk (G.D.); c.fare12@ 123456imperial.ac.uk (C.F.)
                [2 ]Protein Crystallography Facility, Centre for Structural Biology, Flowers Building, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK; rhodri.morgan@ 123456imperial.ac.uk
                [3 ]Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; takanori.nakane@ 123456bs.s.u-tokyo.ac.jp
                [4 ]Quantum Optics and Laser Science Group, Blackett Laboratory, Imperial College, London SW7 2AZ, UK; a.sanchez-gonzalez13@ 123456imperial.ac.uk (A.S.-G.); allan.s.johnson@ 123456gmail.com (A.S.J.); j.marangos@ 123456imperial.ac.uk (J.P.M.)
                [5 ]Diamond Light Source Ltd., Diamond House, Harwell Science & Innovation Campus, Didcot OX11 0DE, UK; ann.fitzpatrick@ 123456diamond.ac.uk
                [6 ]LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Rd., Menlo Park, CA 94025, USA; yoon82@ 123456stanford.edu (C.H.Y.); mhunter2@ 123456slac.stanford.edu (M.S.H.); deponte@ 123456slac.stanford.edu (D.P.D.); sboutet@ 123456slac.stanford.edu (S.B.)
                [7 ]RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Hyogo 679-5148, Japan; osigeki@ 123456spring8.or.jp (S.O.); rie.tanaka.hw@ 123456riken.jp (R.T.); tono@ 123456spring8.or.jp (K.T.); s.iwata@ 123456spring8.or.jp (S.I.)
                [8 ]Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Hyogo 679-5198, Japan
                [9 ]Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
                Author notes
                [* ]Correspondence: j.vanthor@ 123456imperial.ac.uk ; Tel.: +44-207-594-5071
                Author information
                https://orcid.org/0000-0003-2697-2767
                https://orcid.org/0000-0002-4411-6131
                https://orcid.org/0000-0002-0711-708X
                https://orcid.org/0000-0003-1482-1662
                Article
                ijms-18-01918
                10.3390/ijms18091918
                5618567
                28880248
                75401329-2fbd-46c4-b9d6-12a3d5586aa0
                © 2017 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 14 August 2017
                : 02 September 2017
                Categories
                Article

                Molecular biology
                xfel,sfx,rsfp,skylan-ns,sacla,lcls
                Molecular biology
                xfel, sfx, rsfp, skylan-ns, sacla, lcls

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