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Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

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Abstract

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

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Figure 1: Experimental schemes for the time-resolved serial femtosecond crystallography experiments on photosystem II.
Figure 2: Overall structure and omit map electron density of photosystem II.
Figure 3: The OEC simulated annealed omit maps.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structure factors and coordinates have been deposited in the Protein Data Bank and accession codes for S1 and putative S3 states are 4PBU and 4Q54, respectively.

Change history

  • 10 September 2014

    Minor changes were made to Fig. 3c labelling.

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Acknowledgements

Experiments were carried out at the Linac Coherent Light Source (LCLS), a national user facility operated by Stanford University on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences (OBES). This work was supported by the following agencies: the Center for Bio-Inspired Solar Fuel Production, an Energy Frontier Research Center funded by the DOE, Office of Basic Energy Sciences (award DE-SC0001016), the National Institutes of Health (award 1R01GM095583), the US National Science Foundation (award MCB-1021557 and MCB-1120997), the DFG Clusters of Excellence ‘Inflammation at Interfaces’ (EXC 306) and the ‘Center for Ultrafast Imaging’; the Deutsche Forschungsgemeinschaft (DFG); the Max Planck Society, the Atomic, Molecular and Optical Sciences Program; Chemical Sciences Geosciences and Biosciences Division, DOE OBES (M.J.B.) and the SLAC LDRD program (M.J.B., H.L.); the US DOE through Lawrence Livermore National Laboratory under the contract DE-AC52-07NA27344 and supported by the UCOP Lab Fee Program (award no. 118036) and the LLNL LDRD program (12-ERD-031); the Hamburg Ministry of Science and Research and Joachim Herz Stiftung as part of the Hamburg Initiative for Excellence in Research. The research at Purdue University was supported by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences DE-FG02-12ER16340 (Y.P.) and the National Science Foundation Graduate Research Fellowship under Grant 0833366 (K.M.D.). We also want to thank the National Science Foundation for providing funding for the publication of this work through the BioFEL Science Technology Center (award 1231306). We thank H. Isobe, M. Shoji, S. Yamanaka, Y. Umena, K. Kawakami, N. Kamiya, J. R. Shen and K. Yamaguchi for permission to show a section of Fig. 6 of their publication ref. 4 in Fig. 3d of this publication. We thank R. Neutze and his team for support and discussions during joint beamtime for the PSII project and his projects on time-resolved wide-angle scattering studies. We thank A. T. Brunger for discussions concerning data analysis. We thank T. Terwilliger for support with parameter setting of phenix.autobuild program for the SA-omit maps. We also wish to thank R. Burnap for discussions concerning interpretation of results of ligand mutagenesis. We thank J. D. Zook for his contributions concerning plastoquinone quantification. We thank M. Zhu for helping to create high resolution figures for this publication. We thank Raytheon for support of our studies by providing night-vision devices.

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Authors and Affiliations

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Contributions

C.K., I.G., R.F., M.S.H., R.L.S., A.R., K.S., G.J.W., S. Boutet, H.N.C., U.W., R.B.D., M.F., J.C.H.S. and P.F. contributed to the design of the experiment; C.K., I.G., K.N.R., J.-H.Y., D.E.C., B.R., C.E.C. and S.R.-C. worked on cell growth and photosystem II isolation; J.J.B., T.A.M. and A.L.M. worked on plastoquinone synthesis; C.K., I.G., K.N.R., D.E.C., B.R. and J.J.B. worked on biochemical and biophysical characterization of the photosystem II samples; C.K., K.M.D., L.Y. and Y.P. worked on EPR experiments to confirm the S3 population; C.K., I.G., M.S.H., D.E.C. and P.F. developed nano/microcrystallization conditions of photosystem II; C.K., I.G., R.F., K.N.R., M.S.H. and D.E.C. grew crystals of photosystem II; C.K., I.G., R.F., K.N.R., J.-H.Y., D.E.C., R.G.S., H. Laksmono, M.J.B., T.-C.C. and P.F. conducted biophysical characterization of photosystem II crystals; C.K., I.G., L.G., M.L., L.L., J. Steinbrener, F.S. and P.F. designed and/or fabricated calibration or backup samples; C.K., I.G., D.W., D.J., D.D., U.W., R.B.D. and P.F. tested and optimized buffer and crystal suspension conditions for injection; D.W., D.J., D.D., R.A.K., U.W. and R.B.D. designed and produced nozzles; R.B.D., U.W., R.L.S., D.W., D.J., D.D., R.A.K., S. Bari. and L.L. developed and operated the injector; R.L.S., J. Steinbrener and L.L. developed and operated the sample delivery system and the anti-settling device; S. Boutet, M.M. and G.J.W. developed diffraction instrumentation; M.M., M.S., G.J.W. and S. Boutet set up and operated the CXI beamline; M.S.H., R.A.K., D.M., S. Boutet, M.F. and P.F. designed and optimized the laser excitation scheme and aligned the lasers; C.K., S. Basu., I.G., R.F., N.A.Z., M.S.H., R.L.S., T.A.W., D.W., D.J., D.E.C., H.F., H. Laskmono, H. Liu, A.B., A.L.A., D.D., R.A.K., S. Bari., K.R.B., M.J.B., T.-C.C., L.G., S.K., C.C., M.L., M.M., K.N., M.S., J. Steinbrener, F.S., C.Y., G.J.W., S. Boutet, H.N.C., U.W., R.B.D., M.F., J.C.H.S. and P.F. collected X-ray diffraction data at the CXI beamline; S. Basu, R.F., N.A.Z., T.A.W., H. Liu, A.B., A.L.A., R.A.K., K.R.B., S.K., K.N., L.G., C.Y., J.C.H.S. and P.F. analysed the femtosecond crystallography X-ray diffraction data; T.A.W., A.B., A.L.A., R.A.K. and H.N.C. developed the data evaluation and/or hit finding programs; S. Basu, R.F. and N.A.Z. merged the 3D data; S. Basu and R.F. refined the structure and calculated the electron density maps; S. Basu, R.F., N.A.Z. and P.F. designed and made the figures; R.L.S., T.A.W., D.W., D.J., R.L.S., A.B., A.L.A., A.R., K.S., S.M., A.V.M., S.P.H.-R., R.G.S., H.N.C., U.W., R.B.D., M.F., J.C.H.S., T.A.M. and A.L.M. contributed to the writing of the manuscript with discussion, comments or edits; C.K., S. Basu, R.F., N.A.Z., K.N.R., H.N.C., M.F., J.C.H.S. and P.F. contributed to the interpretation of the results; C.K., S. Basu, I.G., R.F., N.A.Z., K.N.R., C.E.C., H.N.C., U.W., R.B.D., M.F., S.R.-C., J.C.H.S. and P.F. wrote and edited the manuscript with discussion and input from all authors.

Corresponding author

Correspondence to Petra Fromme.

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Extended data figures and tables

Extended Data Figure 1 Photosystem II crystal growth and characterization.

a, Scheme of free interface diffusion enhanced sedimentation method for growth of photosystem II nano/microcrystals. b, Large photosystem II crystals (average size 300 μm) suitable for X-ray data collection at synchrotron sources. c, Optical image of nano/microcrystals of photosystem II grown by free interface diffusion used for the TR-SFX data collection at LCLS. The crystallinity must be confirmed by other methods such as SONICC (see (e) for the SONICC image of the crystals) because nanocrystals look similar to amorphous precipitate. d, DLS results of the crystals shown in c indicate an average Stokes radius of 1 µm. e, SONICC image of the photosystem II microcrystals shown in c. f, g, Panels showing the EPR analysis of S-states yield of PSII after double-excitation. f, X-band EPR spectra (10 K) of photosystem II protein solution used for crystallization exposed to 0 (dark adapted sample, no flash NF), one (1F) or two (2F) saturating laser excitation flashes at room temperature. The samples were flash frozen after illumination. For comparison we also show the EPR spectra of dark adapted photosystem II subjected to continuous illumination at 190 K (NF, illu). At low temperature, the S-state cycle stops in the S2 state which means that this conditions corresponds to the maximal yield of multi-line signal. Three individual samples of each type were analysed and the same MLS intensities were consistently found for similar samples. g, Fit of the quantified S2 state multiline signal (MLS) oscillations to the Kok model of the S-state transition cycle31. Please note that the MLS yield after the second and third flash is nearly constant in the measurements, whereas the fit predicts a decline after the third flash. This is expected as we have not added quinones or artificial electron acceptors to the sample, so that there is no terminal electron acceptor present after PQH2 has left the QB binding site after the second flash.

Extended Data Figure 2 Background corrected diffraction pattern of a photosystem II microcrystal.

a, b, From the dark (S1) data set (a) and the double-flash data set (b) collected at the CXI instrument at LCLS. The resolution is indicated by red and yellow rings corresponding to resolution shells in Å 10, 9, 8 (red), 7 (orange), 6, 5, 4 (yellow). The right panel shows an enlarged view of the diffraction patterns (see blue box).

Extended Data Figure 3 Distribution of photosystem II unit cell constants of 4 different femtosecond crystallography data sets.

Row 1 (top row) shows unit cell constants of the dark data set (S1 state) collected at the CXI instrument in January 2012 (experiment (A)). Row 2 shows unit cell constants of the double-flash data set (putative S3 state) collected at the CXI instrument in January 2012 (experiment (A)). Row 3 shows unit cell constants of the dark data set (S1 state) collected at the CXI instrument in June 2012 (experiment (B)) (quinone PQdecyl was added to these crystals to allow replacement of the quinone for triple excitation). Row 4 shows unit cell constants of the triple-flash data set (putative S4 state) collected at the CXI instrument in June 2012 (experiment (B)). The comparison of unit cell constants shows that significant changes in the unit cell constants are observed after double-flash excitation of photosystem II. These changes are fully reversed when photosystem II is excited by three laser flashes. Although the number of indexed patterns currently available does not yet allow for the determination of an accurate structure of the PSII after triple excitation, the data allows extraction of information on the hit rates, indexing rates and unit cell constants, showing that the unit cell constants are identical for the dark S1 and triple-flash state.

Extended Data Figure 4 Rsplit as a function of resolution bins and number of indexed patterns.

a, Rsplit as a function of the resolution shell (in total 20 bins) for dark state data (blue) and double-pumped state data (red). b, Rsplit as a function of resolution bins for dark S1 state, Rsplit decreases indicating better data quality with increase in number of indexed patterns from 3,300 to 34,000 images. c, Rsplit as a function of resolution bins for the dark and double-flash states, the Rsplit decreases indicating better quality with increase in number of indexed patterns from 1,800 to 18,800 images.

Extended Data Figure 5 The arrangement of the transmembrane helices in the photosystem II dimer.

a, b, An overview of the arrangement of transmembrane helices in photosystem II. The protein subunits are named according to their genes so PsbA is subunit A, PsbB is subunit B, etc. The identification of the location of subunits with more than one transmembrane helix is facilitated by ovals, which are labelled using the same colour code as the corresponding subunit. a, Top view from the stromal side of the arrangement of transmembrane helices in the middle of the membrane. The assignment of helices to different protein subunits is based on the structural assignments of ref. 6. The 5 transmembrane helices of the core subunits of the reaction centre are PsbA (blue) and PsbB (red). b, The picture shows the omit maps (2Fo-Fc) of the dark and double-flash states at the contour level of 1.5 σ in the same view direction as shown in Extended Data Fig. 5a. cf, These panels show that most of the alpha helices in the middle of the membrane are well matched between the dark and double-flash states, in the reaction centre core (PsbA and PsbB) as well as in the peripheral parts of photosystem II (for example PsbZ). The view and colour coding of helices are the same as in Extended Data Fig. 5a. c, d, Omit maps of the dark (green) and double-flash (white) states of PSII show a cut through the central region of photosystem II at 1.5 σ. e, The superposition of omit maps indicates a good general overlay of the transmembrane helices and the lumenal loop regions in the two omit maps featuring the reaction centre core subunits PsbA (blue) and PsbB (red) as well as the peripheral subunit X (cyan), and the subunits M (pink) and L (grey) in the dimerization domain of the photosystem II dimer. The electron density is shown at the contour level of 1.5 σ. f, The structural model is also shown with same colour codes as in Extended Data Fig. 5a.

Extended Data Figure 6 Omit map of the dark and double-flash states of the most peripheral photosystem II membrane integral subunits and the chlorophylls of the primary electron donor P680.

a–d, This picture features the peripheral subunits PsbZ (grey-green), PsbK (brown), PsaY (grey) and the core-antenna protein CP43 (PsbC) (cyan). The omit electron density map at the contour level of 1.5 σ for the dark (S1) state is depicted in green (a) and the double-flash (putative S3) state is depicted in white (b). c, The overlay of the two omit maps is shown at the contour level of 1.5 σ. The globular densities between PsbC and PsbK correspond to antenna chlorophylls. The figure shows that even the electron density for the two most peripheral helices that belong to subunit PsbZ are well defined. We also note the good match of the strongly kinked helix of PsbK between the S1 and S3-state electron density maps. d, The subunits are labelled according to their genes in the view of the structural model. eh, The figure features the surroundings of the two chlorophylls of P680 and the accessory chlorophyll of the active electron transfer branch of photosystem II (see Fig. 2c). The omit electron density map at the contour level of 1.5 σ for the dark (S1) state is depicted in green (e) and the double-flash (putative S3) state is depicted in white (f). g, The figure also shows the overlay of the two omit maps at the contour level of 1.5 σ. h, Model of the chlorophylls of the primary electron donor P680 without electron density map.

Extended Data Figure 7 The electron acceptor side of photosystem II.

Omit map electron density and structural model of the dark and double-flash state of photosystem II, the view from the stromal side onto the membrane plane. a–d, The loops that coordinate the non-haem iron and cover the quinone binding sites looking from the stromal side onto the membrane plane. The omit electron density map at the contour level of 1.5 σ for the dark (S1) state is depicted in green (a) and the double-flash (putative S3) state is depicted in white (b). c, The overlay of the two omit maps at 1.5 σ. d, The structural model indicates the positions of PQA and PQB as well as the non-haem iron located below the loops. We note that the electron densities of the loop regions at the electron acceptor side show significant differences between the dark and the double-flash states. The electron density of both states may suggest a conformation of the loops that could differ in their backbone trace from the model derived from the 1.9 Å structure from ref. 6. eh, The side view of the acceptor side of photosystem II along the plane in the membrane. The omit electron density map at the contour level of 1.5 σ for the dark (S1) state is depicted in green (e) and the double-flash (putative S3) state is depicted in white (f). g, The overlay of the two omit maps featuring the changes in the position of the non-haem iron and loop regions at the contour level of 1.5 σ. h, Model of the electron acceptor side of photosystem II. The protein subunits are colour coded as in Extended Data Fig. 5a of the main text; the non-haem iron is depicted as a red sphere. The tightly bound plastoquinone PQA is shown in white, the mobile plastoquinone PQB is depicted in cyan.

Extended Data Figure 8 Simulated annealed omit map of the Mn4CaO5 cluster of photosystem II.

The electron density of the dark state of photosystem II. This figure shows the superimposed SA-omit maps for the dark (S1) (blue) state of the Mn4CaO5 cluster. We use a different colour scheme for the SA-omit maps and the ‘regular 2Fo-Fc’ omits maps to allow the reader a better orientation of the type of map shown in each figure. The electron density is shown at the contour level of 3.0 σ to ensure that it solely features the metal Mn4CaO5 cluster. The X-ray structure at 1.9 Å from ref. 6 is placed inside the SA-omit map for comparison. The nomenclature for the Mn atoms proposed in ref. 6 is used for the colour-coding of the individual Mn atoms in the cluster. The Mn ions that form the distorted Mn3OxCa cubane (Mn1, Mn2 and Mn3) are depicted in light pink, while Mn4 (violet) (referred to as the dangler Mn) is located outside the cubane. This figure shows that the dangler Mn sticks out of the SA-omit map electron density, which indicates that this Mn atom may be located in closer proximity to the Mn3OxCa cubane in the dark S1 state that is not influenced by X-ray damage.

Extended Data Table 1 Statistics of the femtosecond crystallography X-ray diffraction data sets
Extended Data Table 2 Data statistic comparison of hit and indexing rates as well as the unit cell constants from 4 different data sets collected on photosystem II crystals

Supplementary information

Graphic representation of the structure factor amplitudes for the dark data set.

Graphic representation of the structure factor amplitudes for the dark data set. The video shows the structure factor amplitudes from photosystem II nanocrystal SFX data collected in the dark at 5.0 Å , representing the dark S1 state of the oxygen evolving complex. The graphic representation was generated using the CrystFEL suite17. (MOV 24087 kb)

Graphic representation of the structure factor amplitudes for the double-flash data set.

The video shows the structure factor amplitudes from photosystem II nanocrystal SFX data collected at 5.5 Å from the double flash state, representing the putative S3 state of the oxygen evolving complex. The graphic representation was generated using the CrystFEL suite17. (MOV 23940 kb)

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Kupitz, C., Basu, S., Grotjohann, I. et al. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser. Nature 513, 261–265 (2014). https://doi.org/10.1038/nature13453

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