Abstract

The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase5 the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing6,7.

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Data deposits

The coordinates of the atomic model obtained by molecular replacement from the Bragg peaks alone, and with the inclusion of continuous diffraction, have been deposited into the Protein Data Bank under accession codes 5E7C and 5E79, respectively.

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Acknowledgements

We acknowledge support of the Helmholtz Association through project-oriented funds and the Virtual Institute “Dynamic Pathways in Multidimensional Landscapes”; the DFG through the Gottfried Wilhelm Leibniz Program; the European Research Council under the European Union’s Seventh Framework Programme ERC Synergy Grant 609920 “AXSIS” and Marie Curie FP7-PEOPLE-2011-ITN Grant 317079 “Nanomem”; the BMBF through Project 05E13GU1; the Graduate College “GRK 1355” at the University of Hamburg, the International Max Planck Research School UFAST, the BioXFEL Science Technology Center (award 1231306); and the US National Institutes of Health (NIH), National Institute of General Medical Sciences grants R01 GM095583, U54 GM094599, and R01 GM097463. Parts of the sample injector used at the Linac Coherent Light Source (LCLS) for this research was funded by the NIH, P41GM103393, formerly P41RR001209. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-AC02-76SF00515.

Author information

Affiliations

  1. Center for Free-Electron Laser Science, DESY, 22607 Hamburg, Germany

    • Kartik Ayyer
    • , Oleksandr M. Yefanov
    • , Lorenzo Galli
    • , Valerio Mariani
    • , Katerina Dörner
    • , Paulraj Lourdu Xavier
    • , Kenneth R. Beyerlein
    • , Thomas A. White
    • , Anton Barty
    •  & Henry N. Chapman
  2. Department of Physics, University of Hamburg, 22761 Hamburg, Germany

    • Dominik Oberthür
    • , Lorenzo Galli
    • , Markus Metz
    • , Paulraj Lourdu Xavier
    •  & Henry N. Chapman
  3. School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, USA

    • Shatabdi Roy-Chowdhury
    • , Shibom Basu
    • , Jesse Coe
    • , Chelsie E. Conrad
    • , Raimund Fromme
    • , Alexander Schaffer
    • , Katerina Dörner
    • , Christopher Kupitz
    • , Jay-How Yang
    •  & Petra Fromme
  4. Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA

    • Shatabdi Roy-Chowdhury
    • , Shibom Basu
    • , Jesse Coe
    • , Chelsie E. Conrad
    • , Raimund Fromme
    • , Alexander Schaffer
    • , Daniel James
    • , Garrett Nelson
    • , John C. H. Spence
    • , Uwe Weierstall
    • , Jay-How Yang
    • , Yun Zhao
    •  & Petra Fromme
  5. Department of Physics, Arizona State University, Tempe, Arizona 85287, USA

    • Daniel James
    • , Garrett Nelson
    • , John C. H. Spence
    • , Uwe Weierstall
    •  & Yun Zhao
  6. Physics Department, University of Wisconsin, Milwaukee, Wisconsin 53211, USA

    • Christopher Kupitz
    •  & Marius Schmidt
  7. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, GR-70013 Crete, Greece

    • Iosifina Sarrou
  8. Linac Coherent Light Source, Stanford Linear Accelerator Center (SLAC), National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA

    • Mengning Liang
    • , Andrew Aquila
    • , Mark S. Hunter
    • , Joseph S. Robinson
    • , Jason E. Koglin
    •  & Sébastien Boutet
  9. Centre for Ultrafast Imaging, 22607 Hamburg, Germany

    • Henry N. Chapman

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Contributions

H.N.C. conceived the idea of molecular imaging using imperfect crystals, and developed it with K.A., A.B., O.M.Y., D.O., V.M., K.R.B. and L.G. K.A., A.B., O.M.Y., D.O., L.G. and V.M. carried out the diffractive imaging analysis. P.F. led the photosystem II project. Serial femtosecond crystallography measurements were led by P.F. and A.B. and performed with S.R.-C., J.C., C.K., D.O., M.M., A.S., K.D., D.J., G.N., U.W., Y.Z. and M.S. M.L., A.A., M.S.H., J.S.R., J.E.K. and S.Bo. set up and operated the CXI instrument. S.Ba., R.F., A.B. and M.M. performed on-site data evaluation at LCLS. S.Ba. and R.F. provided the initial search model and carried out model refinement on the image with D.O. Photosystem II was isolated by S.R.-C., J.C., C.E.C., A.S., C.K., A.S., J.-H.Y. and K.D., from which microcrystals were grown by S.R.-C., J.C., C.E.C., A.S. and C.K. Test experiments on crystals were performed by K.D., I.S., P.L.X. and M.M. H.N.C., A.B., K.A., O.M.Y., D.O., L.G., P.F., J.C.H.S., T.A.W., S.Ba. and S.Bo. wrote the manuscript with improvements from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Henry N. Chapman.

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https://doi.org/10.1038/nature16949

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