Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser Christopher Kupitz1*, Shibom Basu1*, Ingo Grotjohann1, Raimund Fromme1, Nadia A. Zatsepin2, Kimberly N. Rendek1, Mark S. Hunter1,3, Robert L. Shoeman4, Thomas A. White5, Dingjie Wang2, Daniel James2, Jay-How Yang1, Danielle E. Cobb1, Brenda Reeder1, Raymond G. Sierra6, Haiguang Liu2, Anton Barty5, Andrew L. Aquila5,7, Daniel Deponte5,8, Richard A. Kirian2,5, Sadia Bari9,10, Jesse J. Bergkamp1, Kenneth R. Beyerlein5, Michael J. Bogan6, Carl Caleman5,11, Tzu-Chiao Chao1,12, Chelsie E. Conrad1, Katherine M. Davis13, Holger Fleckenstein5, Lorenzo Galli5,14, Stefan P. Hau-Riege3, Stephan Kassemeyer4,9, Hartawan Laksmono6, Mengning Liang5, Lukas Lomb4, Stefano Marchesini15, Andrew V. Martin5,16, Marc Messerschmidt8, Despina Milathianaki8, Karol Nass4,5,14, Alexandra Ros1, Shatabdi Roy-Chowdhury1, Kevin Schmidt2, Marvin Seibert8,17, Jan Steinbrener4, Francesco Stellato5, Lifen Yan13, Chunhong Yoon5,7, Thomas A. Moore1, Ana L. Moore1, Yulia Pushkar13, Garth J. Williams8, Sébastien Boutet8, R. Bruce Doak2, Uwe Weierstall2, Matthias Frank3, Henry N. Chapman5,14,18, John C. H. Spence2 & Petra Fromme1** 1 Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA. 2 Department of Physics, Arizona State University, Tempe, Arizona 85287, USA. 3 Lawrence Livermore National Laboratory, Livermore, California 94550, USA. 4 Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany. 5 Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany. 6 Stanford PULSE Institute, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA. 7 European XFEL GmbH, Notkestrasse 85, 22607 Hamburg, Germany. 8 Linac Coherent Light Source, Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA. 9 Max Planck Advanced Study Group, Center for Free-Electron Laser Science (CFEL), Notkestrasse 85, 22607 Hamburg, Germany. 10 Max-Planck-Institut für Kernphysik, Saupfercheckweg 1, 69117 Heidelberg, Germany. 11 Department of Physics and Astronomy, Uppsala University, Regementsvägen 1, SE-752 37 Uppsala, Sweden. 12 University of Regina, 3737 Wascana Pkwy Regina, Saskatchewan S4S 0A2, Canada. 13 Department of Physics, Purdue University, 525 Northwestern Avenue, West Lafayette, Indiana 47907, USA. 14 University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany. Page 1 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 15 Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. 16 Department ARC Centre of Excellence for Coherent X-ray Science, of Physics University of Melbourne, Parkville VIC 3010, Australia. 17 Uppsala University, Sankt Olofsgatan 10B, 753 12 Uppsala, Sweden. 18 Center for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany. *These authors contributed equally to this work. ** Please address all questions and responses to the corresponding author Petra Fromme, pfromme@asu.edu Photosynthesis is the process which plants, algae and some bacteria use to convert sunlight to energy thus sustaining all 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 experiments performed with using a new technique, serial femtosecond crystallography (SFX), a recently developed2 , applied to time-resolved experiments on PSII nano/microcrystals. S from Thermosynechococcus elongatustructures 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 site 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 "outlyingMn" and the cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3,4. This work shows the potential for time-resolved serial femtosecond crystallography (TR-SFX) for investigation of catalytic processes in biomolecules. The first X-ray structure of PSII was determined to a resolution of 3.8 Å in 2001 (ref. 5), revealing the protein’s architecture and the overall shape and location of the OEC. In 2011, Shen and co-workers achieved a breakthrough in the structural elucidation by dramatically improving crystal quality, enabling determination at 1.9 Å resolution6. This structure showed the OEC at Page 2 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 near atomic resolution. However, the OEC was probably affected by X-ray damage, a fundamental problem in X-ray crystallography. The X-ray damage problem may be overcome through the use of SFX2,7–9, an advance enabled by the advent of the X-ray free electron laser (XFEL). In SFX, a stream of microcrystals in their mother liquor is exposed to intense 120 Hz femtosecond XFEL pulses, thereby collecting millions of X-ray diffraction ‘snapshots’ in a time-frame of hours. Each X-ray FEL pulse is so intense that it destroys the sample; however, the pulse duration is so short that diffraction is observed before destruction occurs10. Conventional X-ray structures correspond to a time and spatially averaged representation of biomolecules, leading to a ‘static’ picture. To capture dynamic processes such as water oxidation in PSII, time-resolved X-ray data can be collected using SFX11,12. Conformational changes may be observed at a time-resolution ranging from femtoseconds to microseconds by combining visible laser excitation with the SFX setup and varying time delays between the optical pump and the X-ray probe snapshot. As partial reflections from crystals in random orientations are recorded, many snapshots must be collected for adequate sampling of the full reflections and three-dimensional reconstruction. A time-resolved pump-probe experiment was performed in 2010 using PSI-ferredoxin crystals as a model system, in which changes in diffraction intensities, consistent with a light-induced electron transfer process in the PSIferredoxin complex and dissociation of the PSI-ferredoxin complex were seen11. The catalytic reaction in PSII is a dynamic process. The oxygen evolution reaction is catalysed by the oxygen evolving complex, in which the electrons are extracted from the Oxygen-Evolving Complex (OEC) in four sequential charge separation events through the Sstate cycle (Kok cycle), as shown in Fig. 1a (see ref. 1 for a review). Recently, pump-probe simultaneous SFX diffraction and X-ray emission spectroscopy (XES) was reported to investigate the dark S1 state and the single flash (S2-state) of PSII 13 The XES data show that the electronic structure of the highly radiation sensitive Mn4CaO5 cluster does not change during femtosecond X-ray exposure13. However, the quantity and quality of X-ray diffraction data was insufficient to determine if any structural changes occurred. We report on microsecond time-resolved SFX experiments conducted at the CXI instrument14 at the Linac Coherent Light Source (LCLS)15. The experimental setup is shown in Page 3 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 Fig. 1b, c. We developed a multiple-laser illumination scheme that progressively excites the OEC in dark-adapted PSII nano/micro crystals by two laser pulses from the dark S1 state via the S2 state to the double-flash putative S3 state. Not all PSII centres progress to the next S-state by a single saturating flash which could lead to heterogeneities. Therefore the S-state reached in the double-flash experiment is indicated as ‘putative S3 state’ throughout the manuscript. The diffraction patterns collected from dark and illuminated crystals were sorted into two data sets. Using the ‘hit finding’ program Cheetah, 71,628 PSII diffraction images were identified from the dark diffraction patterns and 63,363 were identified from the double-flash patterns. From these hits, 34,554 dark state patterns and 18,772 double-flash patterns were indexed and merged to reduce all stochastic errors using the CrystFEL software suite16 (see Extended Data Table 2a, b). The data were indexed as orthorhombic, with unit-cell parameters of a = 133 Å, b = 226 Å, and c = 307 Å for the dark state, and a = 136 Å, b = 228 Å, and c = 308 Å for the double-flash state. The distributions of unit cell dimensions are shown in Extended Data Fig. 3 and Extended Data Table 2a, b. The data clearly supports an increase in unit cell dimensions in the double-flash state, with the largest difference detected for the a axis. Two factors may explain the change in unit cell constants, lower indexing rates, and a slight decrease in resolution of diffraction: crystal degradation upon laser illumination or significant structural changes upon the transition from the dark state to the double flash state, which may represent the putative S1toS3 transition. To distinguish between these two possibilities, we collected data with triple-flash excitation of the PSII crystals, where at least part of the PSII centres may have reached the putative transient S4 state. Preliminary data evaluation of the triple-flash data set (that is, putative S4 state) shows similar unit cell dimensions and crystal quality as the dark S1 state (see Extended Data Fig. 3 and Extended Data Table 2a). This suggests that conformational changes induced in PSII by the double-flash excitation (that is, during the putative S1toS3 transition) are reversed after excitation with the third flash (in the putative S3toS4 transition), as discussed in the Supplementary Discussion. Diffraction data from the dark and double-flash states were evaluated to 5 Å and 5.5 Å resolution, respectively; the data refinement statistics are shown in Table 1. Since each diffraction pattern represents a thin cut through reciprocal space by the Ewald sphere, only partial reflections were recorded. A high multiplicity of observations is therefore needed for each Page 4 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 Bragg reflection to obtain full, accurate structure factors. The average multiplicity per reflection was 617 for the dark state data set and 383 for the double-flash data set over the whole resolution range (see Extended Data Tables 1a, b). Extended Data Table 2c shows a comparison of the data statistics of this work with the S1 and S2 data in ref. 13. The data were phased by molecular replacement using a truncated version of the 1.9 Å structure (PDB code 3ARC)6. Rigid body refinement (phenix.refine) was performed for both the dark and double-flash structures (see Supplementary Discussion of Methods for further details on molecular replacement and refinement). To reduce model bias, we calculated omit maps and simulated annealed maps (SA-omit maps) for the dark and double-flash data, deleting the coordinates of the Mn4CaO5 cluster from the model. Figure 2a–c shows the arrangement of protein subunits and cofactors of photosystem II, including the electron transport chain. The comparison of the electron density maps for the dark state (green) and the double-flash state (white) at a contour level of 1.5  is shown in Fig. 2d–f. Both maps show clear electron densities for the transmembrane helices as well as loops and cofactors. Additional electron density maps for representative structural elements of PSII are shown in Extended Data Figs 5, 6, 7 and 8. Overall, the protein fits into the electron densities for the dark and double-flash states and matches the high resolution structural model. However, differences appear in regions of the Mn4CaO5 cluster and the acceptor side, where the quinones and the non-haem iron are located. Determining the significance of these changes and their correlations is complicated by the low resolution of the data. Figure 2g–i shows detailed views of the loops at the acceptor side of PSII. The quinones are not visible at the current resolution of 5 Å. The maps indicate differences between the electron densities of the dark and double-flash states in the loop regions and also in the position of the non-haem iron that is coordinated by the loops. We now focus on the structure in the undamaged dark S1 state of the metal cluster in the OEC and the potential light-induced structural changes that may occur during the S-state transition. Extended Data Fig. 8 shows the SA-omit map of the OEC in the dark S1 state for the Mn cluster in PSII with the 1.9 Å X-ray structure in ref. 6. Interestingly, the electron-density map of the ‘dangler’ Mn atom from the 1.9 Å structure is located outside the dark S1 state electron density, a feature also visible in the electron density map of ref. 13 These structural observations Page 5 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 are consistent with spectroscopic results, which indicate that the distance between the dangler Mn and the Mn3OxCa distorted cubane is indeed shorter in the dark S1 state than in the 1.9 Å structure based on the synchrotron data, which might be influenced by X-ray induced reduction of the Mn ions in the metal cluster17,18. This shorter distance is in agreement with density function theory (DFT) studies4,17,19,4 based on the 1.9 Å structure of PSII6, however, the current resolution limit of 5 Å does not allow a quantitative assessment. The mechanism for water splitting has been intensely debated and many models have been proposed. The recent 1.9 Å X-ray structure6 formed the basis for more detailed theoretical studies of the process, yet the proposed mechanisms differ4,19–21. Based on our time-resolved-SFX (TR-SFX) structural data, we looked for differences between the electron-density maps of the OEC, derived from the dark and the double-flash data sets. Figure 3a, b shows the SA-omit maps calculated for dark (blue) and double-flash state (yellow) and compared with the model of the metal cluster from the 1.9 Å structure6 (Fig. 3c). The Mn4CaO5 cluster was omitted from the model for the calculation of the SA-omit map, which includes annealing at a virtual temperature of 5000 K to minimize phase bias. The SA-omit electron densities of the dark and double-flash states differ in the shape and position, as well as in the protein environment, of the Mn4CaO5 cluster. The dark state simulated-annealed (SA)-omit electron density for the OEC protein environment matches the model of the 1.9 Å structure6, whereas the SA-omit map of the doubleflash state differs significantly. Any interpretation of changes in the protein environment of the OEC is highly speculative at a resolution of 5 Å and heterogeneities in the S-state transitions. However, the SA-omit map of the double-flash state is suggestive of conformational changes which may indicate of a movement of the CD loop (including the ligand D170) away from the cluster. If confirmed at higher resolution, this could explain mutagenesis studies that questioned D170 as a ligand in the higher S-states22. Furthermore, in the double flash state, the electron density of the metal cluster extends and shows a new connection to the AB loop at site where D61 is located. Although D61 only serves as a second sphere ligand in the 1.9 Å crystal structure6, mutagenesis studies indicated an important role in the water oxidation process as the S2 to S3 transition is blocked in D61 mutants. The change in the electron-density of the OEC is suggestive of an increase in the distance between the cubane and the ‘dangler’ Mn and distortion in the cubane in the double-flash state. The observed electron densities (Fig. 3a, b) of the dark state and double flash state are consistent Page 6 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 with conformational changes predicted in a recent DFT study of the S3 state in ref. 4, shown in Fig. 3d. The increased distance between the cubane and ‘dangler’ Mn could allow the second ‘substrate’ water molecule to bind between the Mn3OxCa cubane and the dangler Mn in S2 to S3 state transition. It was shown by EXAFS that the Mn–Ca2+ distances in the Mn3OxCa cubane shrink in the S3 state23. Although the Jahn-Teller effect extends the distances between metals in the lower S-states of the OEC (Mn oxidation states +II, +III and +IV), a shrinking of the Mn3OxCa cubane is predicted in the S3 state when all 4 Mn in the OEC have reached the oxidation state +IV. A comparison of the electron density in the dark and the double-flash states may indeed suggest an overall decrease in the dimension of the Mn3OxCa cubane in the doubleflash state, which is in good agreement with the proposed S3 state extended X-ray absorption fine structure (EXAFS) and XES models24 (more detail in the Supplementary Discussion). The consistency of spectroscopy and DFT studies with our observations may provide preliminary indications that a significant fraction of the OEC centres in our crystals have reached the S3 state in the double flash experiment. Our time-resolved SFX study captures the image of PSII after it has been excited by 2 saturating flashes and provides experimental evidence for structural changes occurring in the putative S3 state of the OEC, accompanied by structural changes at the acceptor side of PSII. As the resolution is limited to 5 Å, the interpretation of the changes observed is preliminary. This work is a proof-of-principle that time-resolved SFX can unravel conformational changes at moderate resolution, and lays the foundation for the high resolution analysis of PSII at all stages of the water oxidation process in the future. To unlock the secrets of the water-splitting mechanism by TR-SFX at atomic detail, the resolution must be further improved and structures must be determined from all the S-states with multiple time delays. METHODS SUMMARY Here, we describe microsecond time resolution optical pump/X-ray probe SFX experiments on PSII nano/micro crystals, to study conformational changes in PSII in the transition from the dark to the double-flash state of PSII, where structures were determined at 5 and 5.5 Å resolution respectively. Nanocrystal growth for SFX was performed using a free-interface diffusion technique (see Extended Data Fig. 1a–e). The size and crystallinity of the samples were monitored by dynamic light scattering (DLS) and second order non-linear imaging of chiral Page 7 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 crystals (SONICC)25. Time-resolved SFX data were collected from PSII crystals delivered to the X-ray FEL interaction region at room temperature in a liquid jet26, The crystals were progressed along the S-state cycle27 from the dark S1 to the putative S3 state by two saturating laser flashes before the structure was probed by interaction with the X-rays flashes (see Fig. 1b, c and Supplementary Discussion of Methods for details). The structure factors and coordinates have been deposited in the Protein Data Bank and accession codes for S1and putative S3 states are 4PBU and 4Q54 respectively. Received 26 November 2013; accepted 4 May 2014; doi:10.1038/nature13453 Published online 9 July 2014. <jrn>1. Renger, G. Mechanism of light induced water splitting in photosystem II of oxygen evolving photosynthetic organisms. Biochim. Biophys. Acta 1817, 1164–1176 (2012). Medline CrossRef</jrn> <jrn>2. Chapman, H. N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73– 77 (2011). 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High-resolution protein structure determination by serial femtosecond crystallography. Science 337, 362–364 (2012). Medline CrossRef</jrn> Page 8 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 <jrn>8. Redecke, L. et al. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339, 227–230 (2013). Medline CrossRef</jrn> <jrn>9. Spence, J. C. H., Weierstall, U. & Chapman, H. N. X-ray lasers for structural and dynamic biology. Rep. Prog. Phys. 75, 102601 (2012). Medline</jrn> <jrn>10. Barty, A. et al. Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements. Nature Photon. 6, 35–40 (2012). Medline CrossRef</jrn> <jrn>11. Aquila, A. et al. Time-resolved protein nanocrystallography using an X-ray free- electron laser. Opt. Express 20, 2706–2716 (2012). Medline CrossRef</jrn> <jrn>12. Neutze, R. & Moffat, K. 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M., Kosheleva, I., Henning, R. W., Seidler, G. T. & Pushkar, Y. Kinetic modeling of the X-ray-induced damage to a metalloprotein. J. Phys. Chem. B 117, 9161– 9169 (2013). Medline CrossRef</jrn> Page 9 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 <jrn>19. Ames, W. et al. Theoretical evaluation of structural models of the S2 state in the oxygen evolving complex of photosystem II: protonation states and magnetic interactions. J. Am. Chem. Soc. 133, 19743–19757 (2011). Medline CrossRef</jrn> <jrn>20. Siegbahn, P. E. Water oxidation mechanism in photosystem II, including oxidations, proton release pathways, O–O bond formation and O2 release. Biochim. Biophys. Acta 1827, 1003–1019 (2013). Medline CrossRef</jrn> <jrn>21. Rivalta, I., Brudvig, G. W. & Batista, V. S. Oxomanganese complexes for natural and artificial photosynthesis. Curr. Opin. Chem. Biol. 16, 11–18 (2012). Medline CrossRef</jrn> <jrn>22. Debus, R. J., Strickler, M. 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Medline CrossRef</jrn> Supplementary Information is available in the online version of the paper. 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 DESC0001016), 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. 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. Author Contributions C.K., I.G., R.F., M.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 Page 11 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 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 S 3 population; C.K., I.G., M.H., D.E.C. and P.F. developed nano/micro crystallization conditions of photosystem II; C.K., I.G., R.F., K.N.R., M.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.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.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., 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 Author Information The structure factors and coordinates have been deposited in the Protein Data Bank and accession codes for S1and putative S3 states are 4PBU and 4Q54, respectively. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to P.F. (pfromme@asu.edu). Figure 1 Experimental schemes for the time-resolved serial femtosecond crystallography experiments on photosystem II. a, S-state scheme of the oxygen-evolving complex depicting changes the oxidation state of the 4 manganese ions of the Mn4CaO5 cluster in the S-state cycle and indicating the reduction of the plastoquinone (PQ) to plastoquinol (PQH2) in the QB site. b, Experimental setup. The crystal-steam of photosystem II, was exposed to two subsequent optical Page 12 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 laser pulses at 527 nm before interacting with the femtosecond X-ray FEL pulses. With a FEL frequency of 120 Hz and triggering of the laser at 60 Hz, X-ray diffraction patterns from crystals in the dark state and ‘light’ double-flash state alternate. c, Laser excitation scheme. The first 527 nm laser pulse excited the crystals 110 s after the trigger pulse. The delay time between the first and second 527 nm laser pulse was 210 s, with X-ray diffraction data collected 570 s after the second laser pulse. Figure 2 Overall structure and omit map electron density of photosystem II . a, Transmembrane helices and cofactors in photosystem II (stromal view density map). The proteins are named according to their genes and labelled with coloured letters. b, Side view of PSII at its longest axis along the membrane plane. c, Electron transport chain of PSII (P680 (blue), accessory chlorophylls (smudge-green), pheophytins (yellow) and plastoquinones PQA (white) and PQB (cyan)); atoms of the OEC are depicted as spheres (Mn purple, Ca green, O light red). d–f, Omit map electron densities (view as in b) at 1.5  for the dark state (S1) (green) (d), double-flash state (putative S3 state) (white) (e) and overlay of the two omit maps (f). g–i, Omit maps (1.5 ) of the electron acceptor side of photosystem II for the dark (S1) (green) (g), double-flash (putative S3 state) (white) (h) and overlay of the two omit maps (i). Note that changes include a shift of the electron density of the non-h iron. Figure 3 The OEC simulated annealed omit maps. a, b, At 1.5  for dark and double flash states of the Mn4CaO5 cluster of PSII for the dark S1-state (blue) (a) and double-flash, putative S3 state (b) with the 1.9 Å structural model (3ARC) from ref. 6. Mn in the distorted Mn3OxCa cubane (Mn-1 to Mn-3) (light-pink), dangler manganese (Mn-4) (violet), calcium (green) and oxygen (red). c, 1.9 Å crystal structure of the Mn4CaO5 cluster with ligands from ref. 6 (PDB accession code: 3ARC). (d) Proposed model of the S3 state based on DFT calculations by Isobe et al.4 (reproduced with permission of The Royal Society of Chemistry) Larger diversions in the SA-omit map of the double-flash (putative S3 state) include potential movement of the loop connecting transmembrane helices C/D (CD loop) with D170 and AB loop (with D61), and an increase of the distance between the dangler Mn and the Mn3OxCa cubane (violet arrow). Table 1: Statistics of the dark (S1) and double-flash (putative S3) data sets collected at CXI, LCLS. Numbers in parentheses refer to values for the highest resolution shell. Wavelength (Å) Dark data set Double-flash data set 2.05 2.05 Page 13 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 Resolution range (Å) 100.6–5.0 102.3–5.5 Space group P212121 P212121 Unit cell length(Å) 133.3 ± 1.6, 226.3±2.1, 307.1±3.1 136.6 ± 1.5, 228.1 ± 2.1, 308.7 ± 3.8 Total reflections 28,679,554 (1,679,683) 12,476,013 (1,018,721) Unique reflections 40,946 (2,710) 32,066 (2,651) Multiplicity 700.35 (618.0) 388.55 (381.1) Completeness (%) 99.98 (100) 99.88(100) Mean I/σ (I) 10.65 (2.1) 8.03(1.75) CC1/2ǂ 0.914 (0.740) 0.877 (0.635) Rsplit 0.07 (0.37) 0.09 (0.49) Rwork 0.260 (0.3502) 0.280 (0.3820) Rfree 0.262 (0.3434) 0.290 (0.3477) RMS* (bonds) Å 0.039 0.039 RMS* (bonds) deg 3.029 3.029 Number of atoms 49,817 49,817 Protein residues 5,214 5,214 Ramachandran* favoured (%) 97.7 97.7 Ramachandran* outliers (%) 0.2 0.2 Clashscore (Molprobity) 5.5 5.8 Mean B-factor † (Å2) 33.7 33.7 Page 14 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 *It is noted that the values for the RMS for bonds and angles as well as the Ramachandran values are positively biased by the high resolution model 3ARC. † The B-factors were taken from the high resolution model 3ARC and not refined. Page 15 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 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 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. It is of note that 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 multiline 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 cycle30. 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 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 Page 16 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 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 theAhit 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 Fig. 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  in the same view direction as shown in Extended Data Fig. 5a. c–f, 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 Page 17 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 (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 . 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 . 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 (greygreen), PsbK (brown), PsaH (grey) and the core-antenna protein CP43 (PsbC) (cyan). The omit electron density map at the contour level of 1.5  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 . 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. e–h, 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  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 . 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 Page 18 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 omit electron density map at the contour level of 1.5  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 . 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. e–h, 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  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 . 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  to ensure that it solely features the metal Mn4CaO5 cluster. The Xray 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) (named 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 a, The dark (S1) state by resolution bins*. b, Statistics of the femtosecond crystallography X-ray diffraction data set of the double-flash (putative S3) state by resolution bins. * CC1/2 is Pearson’s coefficient calculated as described in ref. 28. Page 19 of 20 Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature13453 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. a, The data sets dark (A) and double-flash (A) were collected at the CXI instrument in January 2012 and may represent the dark S1 state and putative S3 state of photosystem II for which data evaluation and structural changes are discussed in this work. For comparison, the statistics are shown for data sets collected on the dark state S1 and the transient triple-flash state (that is, putative S4 state) at the CXI instrument in June 2012. b, Data statistics for dark (A) separated into runs where the laser was switched off (only dark state) and dark state images from runs where alternate dark and light states patterns were recorded. c, Data statistics from this work and from ref. 13. Comparison of the data evaluation statistics of the dark S1 state and double-flash (putative S3) state data from this work evaluated by the CrystFEL software suite16 along with data from ref. 13 on the dark S1 state and the single excited S2 state evaluated with the software suite cctbx.xfel29. The numbers in brackets refer to the data statistics in the highest resolution shell. Page 20 of 20