Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Time-resolved serial femtosecond crystallography at the European XFEL

Abstract

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The photocycle of PYP in crystals.
Fig. 2: Pulse train structure and laser excitation.
Fig. 3: TR-SFX experiments at LCLS and EuXFEL.
Fig. 4: DED and structures of the chromophore-binding region of PYP.
Fig. 5: Time series of TRX data from 3 ps to 100 ps collected at LCLS, EuXFEL and APS.

Similar content being viewed by others

Data availability

Data has been deposited with the Coherent X-ray Imaging Data Bank73 with CXIDB ID 100. This includes: stream files for all data and for data separated into each time delay, MTZ and PDB files for all time delays, including the dark/reference structures. We have deposited data (mtz-files and structures) for the 10 ps, 30 ps and 80 ps time delays, as well as the dark3 (30 ps) and pure dark reference structures, with the Protein Data Bank, with deposition codes 6P4I, 6P5D, 6P5E, 6P5G and 6P5F, respectively.

Code availability

Linux scripts and Fortran source codes for the calculation of weighted difference maps, extrapolated electron density maps and the integration of negative densities within a spherical volume are included in a demonstration, which is available online as Supplementary Data.

References

  1. Moffat, K. Time-resolved biochemical crystallography: a mechanistic perspective. Chem. Rev. 101, 1569–1581 (2001).

    CAS  PubMed  Google Scholar 

  2. Schmidt, M. Time-resolved macromolecular crystallography at modern X-ray sources. Methods Mol. Biol. 1607, 273–294 (2017).

    CAS  PubMed  Google Scholar 

  3. Aquila, A. et al. Time-resolved protein nanocrystallography using an X-ray free-electron laser. Opt. Express 20, 2706–2716 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Tenboer, J. et al. Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein. Science 346, 1242–1246 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Chapman, H. N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Boutet, S. et al. High-resolution protein structure determination by serial femtosecond crystallography. Science 337, 362–364 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Lomb, L. et al. Radiation damage in protein serial femtosecond crystallography using an X-ray free-electron laser. Phys. Rev. B 84, 214111 (2011).

    Google Scholar 

  8. Nass, K. et al. Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams. J. Synchrotron Radiat. 22, 225–238 (2015).

    CAS  PubMed  Google Scholar 

  9. Suga, M. et al. Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543, 131–135 (2017).

    CAS  PubMed  Google Scholar 

  10. Chreifi, G. et al. Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer. Proc. Natl Acad. Sci. USA 113, 1226–1231 (2016).

    CAS  PubMed  Google Scholar 

  11. Wiedorn, M. O. et al. Megahertz serial crystallography. Nat. Commun. 9, 4025 (2018).

    PubMed  PubMed Central  Google Scholar 

  12. Grünbein, M. L. et al. Megahertz data collection from protein microcrystals at an X-ray free-electron laser. Nat. Commun. 9, 3487 (2018).

    PubMed  PubMed Central  Google Scholar 

  13. Barends, T. R. et al. Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science 350, 445–450 (2015).

    CAS  PubMed  Google Scholar 

  14. Pande, K. et al. Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352, 725–729 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Meyer, T. E., Yakali, E., Cusanovich, M. A. & Tollin, G. Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin. Biochemistry 26, 418–423 (1987).

    CAS  PubMed  Google Scholar 

  16. Genick, U. K. et al. Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science 275, 1471–1475 (1997).

    CAS  PubMed  Google Scholar 

  17. Ihee, H. et al. Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc. Natl Acad. Sci. USA 102, 7145–7150 (2005).

    CAS  PubMed  Google Scholar 

  18. Kort, R. et al. Evidence for transcis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein. FEBS Lett. 382, 73–78 (1996).

    CAS  PubMed  Google Scholar 

  19. Polli, D. et al. Conical intersection dynamics of the primary photoisomerization event in vision. Nature 467, 440–443 (2010).

    CAS  PubMed  Google Scholar 

  20. Mathes, T. et al. Femto- to microsecond photodynamics of an unusual bacteriophytochrome. J. Phys. Chem. Lett. 6, 5 (2014).

    Google Scholar 

  21. Ali, A. M. et al. Optogenetic inhibitor of the transcription factor CREB. Chem. Biol. 22, 1531–1539 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Schotte, F. et al. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc. Natl Acad. Sci. USA 109, 19256–19261 (2012).

    CAS  PubMed  Google Scholar 

  23. Creelman, M., Kumauchi, M., Hoff, W. D. & Mathies, R. A. Chromophore dynamics in the PYP photocycle from femtosecond stimulated Raman spectroscopy. J. Phys. Chem. B 118, 659–667 (2014).

    CAS  PubMed  Google Scholar 

  24. Palmer, G. et al. Pump-probe laser system at the FXE and SPB/SFX instruments of the European X-ray free-electron laser facility. J. Synchrotron Radiat. 26, 328–332 (2019).

    CAS  PubMed  Google Scholar 

  25. Schmidt, M. et al. Protein energy landscapes determined by five-dimensional crystallography. Acta Crystallogr. D 69, 2534–2542 (2013).

    CAS  PubMed  Google Scholar 

  26. Prokhorenko, V. I. et al. Coherent control of retinal isomerization in bacteriorhodopsin. Science 313, 1257–1261 (2006).

    CAS  PubMed  Google Scholar 

  27. Mancuso, A. P. et al. The single particles, clusters and biomolecules and serial femtosecond crystallography instrument of the European XFEL: initial installation. J. Synchrotron Radiat. 26, 660–676 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Allahgholi, A. et al. The adaptive gain integrating pixel detector at the European XFEL. J. Synchrotron Radiat. 26, 74–82 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Stan, C. A. et al. Liquid explosions induced by X-ray laser pulses. Nat. Phys. 12, 966–971 (2016).

    CAS  Google Scholar 

  30. Tripathi, S., Srajer, V., Purwar, N., Henning, R. & Schmidt, M. pH dependence of the photoactive yellow protein photocycle investigated by time-resolved crystallography. Biophys. J. 102, 325–332 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Schmidt, M. in Ultrashort Laser Pulses in Medicine and Biology (eds Braun, M. et al.) 201–241 (Springer, 2008).

  32. Schmidt, M. Time-resolved macromolecular crystallography at pulsed X-ray sources. Int. J. Mol. Sci. 20, 1401 (2019).

    PubMed Central  Google Scholar 

  33. Jung, Y. O. et al. Volume-conserving transcis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography. Nat. Chem. 5, 212–220 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hutchison, C. D. M. & van Thor, J. J. Populations and coherence in femtosecond time resolved X-ray crystallography of the photoactive yellow protein. Int, Rev. Phys. Chem. 36, 117–143 (2017).

    CAS  Google Scholar 

  35. Groenhof, G. et al. Photoactivation of the photoactive yellow protein: why photon absorption triggers a trans-to-cis isomerization of the chromophore in the protein. J. Am. Chem. Soc. 126, 4228–4233 (2004).

    CAS  PubMed  Google Scholar 

  36. Markovitch, O. & Agmon, N. Structure and energetics of the hydronium hydration shells. J. Phys. Chem. A 111, 2253–2256 (2007).

    CAS  PubMed  Google Scholar 

  37. Levantino, M. et al. Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser. Nat. Commun. 6, 6772 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. DePonte, D. P. et al. Gas dynamic virtual nozzle for generation of microscopic droplet streams. J. Phys. D 41, 195505 (2008).

    Google Scholar 

  39. Schmidt, M., Rajagopal, S., Ren, Z. & Moffat, K. Application of singular value decomposition to the analysis of time-resolved macromolecular X-ray data. Biophys. J. 84, 2112–2129 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Rajagopal, S., Schmidt, M., Anderson, S., Ihee, H. & Moffat, K. Analysis of experimental time-resolved crystallographic data by singular value decomposition. Acta Crystallogr. D 60, 860–871 (2004).

    PubMed  Google Scholar 

  41. Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kupitz, C. et al. Structural enzymology using X-ray free electron lasers. Struct. Dyn. 4, 044003 (2017).

    PubMed  Google Scholar 

  43. Olmos, J. L. Jr. et al. Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography. BMC Biol. 16, 59 (2018).

    PubMed  PubMed Central  Google Scholar 

  44. Paul, K. et al. Coherent control of an opsin in living brain tissue. Nat. Phys. 13, 1111–1116 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, J. et al. Time-resolved protein activation by proximal decaging in living systems. Nature 569, 509–513 (2019).

    CAS  PubMed  Google Scholar 

  46. Pandey, S., Bean, R., Sato, T., Mancuso, A. P. & Schmidt, M. Time-resolved serial femtosecond crystallography at the European X-ray free electron laser. Prot. Exch. https://doi.org/10.21203/rs.2.14634/v1 (2019).

  47. Mancuso, A. P., Aquila, A., Borchers, G., Giewekemeyer, K. & Reimers, N. Technical Design Report: Scientific Instrument Single Particles, Clusters, and Biomolecules (SPB) https://doi.org/10.3204/XFEL.EU/TR-2013-004 (XFEL.EU, 2013).

  48. Echelmeier, A. et al. 3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating. J. Appl. Cryst. 52, 997–1008 (2019).

    CAS  Google Scholar 

  49. Hutchison, C. D. M. et al. Photocycle populations with femtosecond excitation of crystalline photoactive yellow protein. Chem. Phys. Lett. 654, 63–71 (2016).

    CAS  Google Scholar 

  50. Nass Kovacs, G. et al. Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin. Nat. Comm. 10, 3177 (2019).

    Google Scholar 

  51. Bean, R. J., Aquila, A., Samoylova, L. & Mancuso, A. P. Design of the mirror optical systems for coherent diffractive imaging at the SPB/SFX instrument of the European XFEL. J. Opt. 18, 074011 (2016).

    Google Scholar 

  52. Greiffenberg, D. The AGIPD detector for the European XFEL. J. Instrum. 7, CO1103 (2012).

    Google Scholar 

  53. Fangohr, H. et al. Data analysis support in Karabo at European XFEL. In Proc. of International Conference on Accelerator and Large Experimental Control Systems (eds Costa, I. et al.) 245–252 (inSPIRE, 2017).

  54. Boukhelef, D., Szuba, J., Wrona, K. & Youngman, C. Software Development for High Speed Data Recording and Processing (JACoW2014).

  55. Kirkwood, H. J. et al. Initial observations of the femtosecond timing jitter at the European XFEL. Opt. Lett. 44, 1650–1653 (2019).

    CAS  PubMed  Google Scholar 

  56. Mariani, V. et al. OnDA: online data analysis and feedback for serial X-ray imaging. J. Appl. Crystallogr. 49, (1073–1080 (2016).

    Google Scholar 

  57. Barty, A. et al. Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data. J. Appl. Crystallogr. 47, 1118–1131 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. White, T. A. et al. Recent developments in CrystFEL. J. Appl. Cryst. 49, 680–689 (2016).

    CAS  Google Scholar 

  59. Gevorkov, Y. et al. XGANDALF – extended gradient descent algorithm for lattice finding. Acta Cryst. Found. Adv. 75, 694–704 (2019).

    CAS  Google Scholar 

  60. Yefanov, O. et al. Accurate determination of segmented X-ray detector geometry. Opt. Express 23, 28459–28470 (2015).

    PubMed  PubMed Central  Google Scholar 

  61. Brehm, W. & Diederichs, K. Breaking the indexing ambiguity in serial crystallography. Acta Crystallogr. D 70, 101–109 (2014).

    CAS  PubMed  Google Scholar 

  62. Glownia, J. M. et al. Time-resolved pump-probe experiments at the LCLS. Opt. Express 18, 17620–17630 (2010).

    CAS  PubMed  Google Scholar 

  63. Harmand, M. et al. Achieving few-femtosecond time-sorting at hard X-ray free-electron lasers. Nat. Photonics 7, 215–218 (2013).

    CAS  Google Scholar 

  64. Bionta, M. R. et al. Spectral encoding of X-ray/optical relative delay. Opt. Express 19, 21855–21865 (2011).

    CAS  PubMed  Google Scholar 

  65. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

    CAS  PubMed  Google Scholar 

  66. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  PubMed  Google Scholar 

  67. Ren, Z. et al. A molecular movie at 1.8 Å resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds. Biochemistry 40, 13788–13801 (2001).

    CAS  PubMed  Google Scholar 

  68. Drenth, J. Principles of Protein X-Ray Crystallography (Springer, 1999)..

  69. Terwilliger, T. C. & Berendzen, J. Bayesian difference refinement. Acta Crystallogr. D 52, 1004–1011 (1996).

    CAS  PubMed  Google Scholar 

  70. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  Google Scholar 

  71. Nogly, P. et al. Retinal isomerization in bacteriorhodopsin captured by a femtosecond X-ray laser. Science 361, eaat0094 (2018).

    PubMed  Google Scholar 

  72. Richards, F. M. & Kundrot, C. E. Identification of structural motifs from protein coordinate data—secondary structure and 1st-level supersecondary structure. Proteins 3, 71–84 (1988).

    CAS  PubMed  Google Scholar 

  73. Maia, F. R. The coherent X-ray imaging data bank. Nat. Methods 9, 854–855 (2012).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge European XFEL in Schenefeld, Germany, for provision of X-ray free-electron laser beamtime at Scientific Instrument SPB/SFX and thank the instrument group and facility staff for their assistance. This work was supported by NSF Science and Technology Centers (grant no. NSF-1231306; Biology with X-ray Lasers) to H.N.C., P.F., A.O., A.R., P.S. and M.S.). CFEL (H.N.C.) is supported by the Gottfried Wilhelm Leibniz Program of the DFG; the project ‘X-probe’ funded by the European Union’s 2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement (no. 637295); the European Research Council, ‘Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)’ (no. ERC-2013-SyG 609920, together with P.F.); and the Human Frontiers Science Program grant (no. RGP0010 2017). P.F. and A.R. acknowledge the support of funding from the Biodesign Center for Applied Structural Discovery at Arizona State University and NSF award (no. 1565180). Funding from the National Institutes of Health (grant nos. R01GM095583 to P.F. and R01GM117342 to M.F.) is also acknowledged.

Author information

Authors and Affiliations

Authors

Contributions

S.P., I.P. and M.S. expressed, purified and crystallized the protein. R.B., T.S., J.B., V.B., M.E., G.G., M.J., Y.K., H.K., A.K., R.L., L.M., T.M., G.P., M.R., A.S., J.S.-D. and A.P.M. operated the SPB/SFX instrument. S.L., J.K., R.S. and H.N.C. provided injector nozzles. J.C.V., C.K., M.H., M.H.A., J.K., F.H.M.K., S.L., V.Maz., D.M., R.S. and A.T. collected the data. S.P., I.P., O.Y., V.Mar., T.A.W., Y.G., A.O., P.S., A.T. and A.B. processed the data. S.P., I.P., P.S., A.O. and M.S. analyzed the data. C.K., M.H.A., R.F. and P.F. logged the experiment. J.C.V., A.E., D.D., D.K. and A.R. conceived and operated the oil co-flow. R.B., T.S., M.F., H.N.C., A.R., A.B., P.F., A.P.M. and M.S. designed the experiment. S.P., S.B., A.B., P.F., A.P.M. and M.S. wrote the manuscript with input from all other authors.

Corresponding author

Correspondence to Marius Schmidt.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Arunima Singh and Allison Doerr were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Fig. 1 Setup of a MHz TR-SFX experiment at the EuXFEL (modified from Wiedorn et al.11).

X-ray pulses arrive in 1.13 MHz bursts which repeat every 100 ms. There are 176 X-ray pulses in the burst. The KB-mirror system focuses the X-ray beam to a 2–3 μm focal spot. The fs-laser delivers 376 kHz pulses (λ=420 nm, blue) synchronized to the X-ray pulses. The laser focus is 42 μm Ø in the X-ray interaction region (dotted circle). The microcrystals are mixed with fluorinated oil and injected by a GDVN. The jet produced by the GDVN, the laser beam as well as the X-ray pulses precisely intersect. The time-resolved diffraction patterns are collected by the AGIPD. Diffraction patterns with common time-delays were separated based on the pulse ID (see also Fig. 2b) and combined to datasets.

Supplementary Fig. 2 Hit and indexing rates.

a, Hit rates (red) and indexing rates (black) with 1.13 MHz X-ray pulse repetition rate. Note, the strong drop of the hit-rate after the first pulse from 2% to 1%. 472,528 total patterns, 41,559 hits and 24,815 indexed patterns were separated on the basis of pulse IDs. From these, hit rates and indexing rates were calculated. b, Hit rates (red) and indexing rates (black) with 564 kHz X-ray pulse repetition. The overall hit rate is about 2%. 52,495,158 total patterns, 304,673 hits and 142,948 indexed patterns were separated on the basis of pulse IDs from which hit rates and indexing rates were calculated. Blue solid line in a and b, X-ray pulse energy (on arbitrary scale). The indexing rate varies only slightly and is about 40% - 60%.

Supplementary Fig. 3 Extrapolated electron density maps (1.5 σ contour level).

a, 3 ps at LCLS (Pande et al.14). b - c, 10 ps, 30 ps and 80 ps at EuXFEL. e, 100 ps at APS (Jung et al. 33). The extrapolated maps were calculated from 13,722, 13,142, 13,014, 12,889 and 13,214 extrapolated structure factors for the 3 ps to 100 ps time delays, respectively.

Supplementary Fig. 4 Excitation and ultrafast displacements in PYP.

a, Structure of PYP. Some important residues in the chromophore (pCA) binding pocket are marked. The M41-71 moiety (residues 41 to 71) is marked in red. Helix H74-88 is marked. b, Dark state spectra of PYP. Black: measured in solution, red: in the crystal. The wavelength at the absorption maximum is marked. Excitation has been achieved with 240 fs laser pulses with λ=420 nm. c, Solid spheres: root mean square displacements of 31 Cα atoms in M41-71 relative to the dark (reference) structure, red spheres: from data measured at EuXFEL. Dashed line: fit by a function consisting of an exponential, a strongly damped, phase shifted cosine function and a straight line as outlined in the text.

Supplementary Fig. 5 Difference distance matrices evaluated for Cα atoms of residues 42 to 93.

The green line denotes the M41-71 moiety. The scale on top is in Å. a - d, Difference distance matrices derived from structures at 10 ps, 30 ps, 80 ps and 100 ps relative to that at 3 ps, respectively. Difference distances are also shown for helix H74-88.

Supplementary Fig. 6 Signal levels in the DED map at the 30 ps delay.

The DED map at 30 ps is overlaid on the entire PYP and contoured from +/- 2σ to +/- 4σ in steps of 0.5σ. Red: negative DED, green: positive DED. The 3σ level, c, is the best compromise to distinguish the signal, for example on the pCA chromophore, from spurious noise features distributed within the protein volume.

Supplementary Fig. 7 Method to determine the factor N and the population transfer (PT).

The factor N has been determined to calculate extrapolated, conventional maps from data collected at various X-ray sources. Black spheres: summed absolute negative DED in a sphere of R = 4 Å centered on the PCA chromophore double bond. Red dotted lines: the more horizontal line follows the initial slope of the data; the second line delineates the constant incline with larger Ns. The Next (in brackets) can be estimated from the intersection of the two lines. a, 3ps data from CXI at LCLS collected with fs laser excitation in the absorption maximum (Pande et al. 14). Factor N = 16, PT = 12.5 %, insert: 1 μs data collected with ns laser excitation. N = 4, and PT = 50% (Tenboer et al.4). b, c, and d, Factors N for the 10 ps, 30 ps, and 80 ps data collected at the EXFEL with fs laser excitation outside the absorption maximum. PT is about 7 % throughout. Insert in d, 100 ps data collected at APS (about 6% PT, Jung et al.33). 13,214, 13,542, 13,722, 13,142, 13,014 and 12,889 observed difference amplitudes are used to determine extrapolated maps for the 100ps, 1 μs, 3ps, 10ps, 30ps and 80ps time delays, respectively.

Supplementary Fig. 8 Observed and calculated difference electron densities (DED) near the pCA chromophore.

Left panels: observed difference electron density (blue: 3 σ, red: -3 σ contour levels). Right panels: calculated difference electron density (blue: 4 σ, red: -4 σ contour levels). Yellow model: structure of the dark (reference) state; blue model: structure at a particular time delay. a, 10 ps; b, 30 ps, c, 80 ps. In panel b pairwise difference density features are marked with α (negative) and β (positive). The feature γ shows the signal caused by the Cys-69 sulfur. The marked DED features can be readily detected at the other time delays. 13,142, 13,014 and 12,889 difference amplitudes were used to calculate the observed DED maps for a, b and c, respectively.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–7.

Reporting Summary

Supplementary Software

demo.tar.zip. Compressed repository containing a demonstration and software for difference map calculation and structure determination. After the TR-SFX experiment datasets of reference (dark) and time-dependent intensities are available. This demonstration guides through the processes of difference map calculation and structure determination from extrapolated electron density maps.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pandey, S., Bean, R., Sato, T. et al. Time-resolved serial femtosecond crystallography at the European XFEL. Nat Methods 17, 73–78 (2020). https://doi.org/10.1038/s41592-019-0628-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-019-0628-z

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing