The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography.

Petra Edlund,Vaibhav Modi,Jason Tenboer,Takanori Nakane,Elin Claesson,Peter Berntsen,Mark S Hunter,Shatabdi Roy-Chowdhury,Emil Gustavsson,Marius Schmidt,Robert Dods,Gerrit Groenhof,James Zook,Emina A Stojković,Keith Moffat,Suraj Pandey,Petra Båth,Oskar Berntsson,Ishwor Poudyal,Anton Barty,Eriko Nango,Tomoyuki Tanaka,Heikki Takala,Petra Fromme,Léocadie Henry,Sebastian Westenhoff,Heli Lehtivuori,Kanupriya Pande,Christopher Kupitz,John C H Spence ,Janne A Ihalainen ,Thomas A White ,Mao Liang ,Jake Koralek ,Matthijs R Panman ,Sébastien Boutet

doi:10.1038/srep35279

Abstract

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.

Highlights

IntroductionResolution in all panels. (a) Superimposed SFX (green) and low temperature (LowT) (grey) structures plotted together with the Fo(SFX)-Fo(LowT) difference map (red: negative, green: positive) at 3.8 σ
Resolution in all panels. (a) Superimposed SFX and low temperature (LowT) structures plotted together with the Fo(SFX)-Fo(LowT) difference map at 3.8 σ
The overall R-factor for difference map is 24.5%. (b) The same representation zoomed into the chromophore region

Summary

Introduction

Resolution in all panels. (a) Superimposed SFX (green) and LowT (grey) structures plotted together with the Fo(SFX)-Fo(LowT) difference map (red: negative, green: positive) at 3.8 σ. Upon light absorption by the chromophore, a sequence of structural changes is initiated, which shuttles the protein from a red light-absorbing state (Pr) to a far-red light-absorbing state (Pfr) or vice versa These structural changes involve at least a Z-to-E isomerization of the C15 =C16 double bond of the chromophore with associated changes of the chromophore-binding pocket[6,7,8,9] and refolding of the so-called PHY-arm[6,7,10,11]. Two Pr-state conformations have been detected for a cyanobacterial phytochrome in solution with NMR spectroscopy[12] This finding is contrasted by the crystal structures of phytochromes, which only show one chromophore conformation[6,7,13,14], at resolutions approaching 1 Å It is not clear whether the crystal packing, or cryogenic temperature at which crystallographic data are recorded, favours only one conformation, or whether the crystal structures truly reflect the solution structures

Methods

Results

Conclusion