Letter | Published:

Femtosecond X-ray protein nanocrystallography

Nature volume 470, pages 7377 (03 February 2011) | Download Citation


X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1,2,3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

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Experiments were carried out at the Linac Coherent Light Source and the Advanced Light Source, both National User Facilities operated respectively by Stanford University and the University of California on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences. We acknowledge support from the DOE through the PULSE Institute at the SLAC National Accelerator Laboratory; the Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344; 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 Hamburg Ministry of Science and Research and the Joachim Herz Stiftung, as part of the Hamburg Initiative for Excellence in Research (LEXI); the Hamburg School for Structure and Dynamics; the Max Planck Society, for funding the development and operation of the CAMP instrument within the ASG at CFEL; the US National Science Foundation (awards 0417142 and MCB-1021557); the US National Institutes of Health (awards 1R01GM095583-01 (ROADMAP) and 1U54GM094625-01 (PSI:Biology)); the Swedish Research Council; the Swedish Foundation for International Cooperation in Research and Higher Education; Stiftelsen Olle Engkvist Byggmästare; the DFG Cluster of Excellence at the Munich Centre for Advanced Photonics; and the CBST at the University of California under cooperative agreement no. PHY 0120999. We acknowledge discussions with M. Rossmann, E. Snell, R. Stroud and A. Brunger, thank B. Hedman, E. Gullikson, F. Filsinger, A. Berg, H. Mahn and C. Kaiser for technical help and thank the staff of the LCLS for their support in carrying out these experiments.

Author information

Author notes

    • Nicola Coppola

    Present address: European XFEL GmbH, Notkestrasse 85, 22607 Hamburg, Germany

    • M. Marvin Seibert

    Present address LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA


  1. Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany

    • Henry N. Chapman
    • , Anton Barty
    • , Thomas A. White
    • , Andrew Aquila
    • , Joachim Schulz
    • , Daniel P. DePonte
    • , Andrew V. Martin
    • , Nicola Coppola
    • , Mengning Liang
    • , Carl Caleman
    • , Lars Gumprecht
    •  & Stephan Stern
  2. University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany

    • Henry N. Chapman
    •  & Karol Nass
  3. Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA

    • Petra Fromme
    • , Mark S. Hunter
    • , Ingo Grotjohann
    •  & Raimund Fromme
  4. Department of Physics, Arizona State University, Tempe, Arizona 85287, USA

    • Richard A. Kirian
    • , Uwe Weierstall
    • , R. Bruce Doak
    • , Kevin E. Schmidt
    • , Xiaoyu Wang
    •  & John C. H. Spence
  5. Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), SE-751 24 Uppsala, Sweden

    • Filipe R. N. C. Maia
    • , Janos Hajdu
    • , Nicusor Timneanu
    • , M. Marvin Seibert
    • , Jakob Andreasson
    • , Andrea Rocker
    • , Olof Jönsson
    •  & Martin Svenda
  6. Max Planck Advanced Study Group, Center for Free-Electron Laser Science, Notkestrasse 85, 22607 Hamburg, Germany

    • Ilme Schlichting
    • , Sascha W. Epp
    • , Daniel Rolles
    • , Artem Rudenko
    • , Lutz Foucar
    • , Benedikt Rudek
    • , Benjamin Erk
    • , Carlo Schmidt
    • , André Hömke
    • , Lothar Strüder
    • , Joachim Ullrich
    •  & Faton Krasniqi
  7. Max-Planck-Institut für Medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany

    • Ilme Schlichting
    • , Lukas Lomb
    • , Robert L. Shoeman
    • , Daniel Rolles
    • , Lutz Foucar
    • , Faton Krasniqi
    • , Mario Bott
    •  & Thomas R. M. Barends
  8. Max-Planck-Institut für Kernphysik, Saupfercheckweg 1, 69117 Heidelberg, Germany

    • Sascha W. Epp
    • , Artem Rudenko
    • , Benedikt Rudek
    • , Benjamin Erk
    • , Carlo Schmidt
    • , André Hömke
    • , Joachim Ullrich
    • , Kai-Uwe Kühnel
    •  & Claus-Dieter Schröter
  9. PNSensor GmbH, Otto-Hahn-Ring 6, 81739 München, Germany

    • Robert Hartmann
    • , Peter Holl
    • , Christian Reich
    •  & Heike Soltau
  10. Max-Planck-Institut Halbleiterlabor, Otto-Hahn-Ring 6, 81739 München, Germany

    • Nils Kimmel
    • , Georg Weidenspointner
    • , Daniel Pietschner
    • , Lothar Strüder
    • , Günter Hauser
    • , Sven Herrmann
    • , Gerhard Schaller
    • , Florian Schopper
    •  & Robert Andritschke
  11. Max-Planck-Institut für Extraterrestrische Physik, Giessenbachstrasse, 85741 Garching, Germany

    • Georg Weidenspointner
  12. Photon Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany

    • Miriam Barthelmess
    • , Saša Bajt
    • , Helmut Hirsemann
    • , Guillaume Potdevin
    • , Heinz Graafsma
    •  & Björn Nilsson
  13. LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA

    • Sébastien Boutet
    • , Jacek Krzywinski
    • , Christoph Bostedt
    • , Marc Messerschmidt
    • , John D. Bozek
    •  & Garth J. Williams
  14. PULSE Institute, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA

    • Michael J. Bogan
    • , Christina Y. Hampton
    • , Raymond G. Sierra
    •  & Dmitri Starodub
  15. Forschungszentrum Jülich, Institut ZEL, 52425 Jülich, Germany

    • Hubert Gorke
  16. Lawrence Livermore National Laboratory, 7000 East Avenue, Mail Stop L-211, Livermore, California 94551, USA

    • Stefan P. Hau-Riege
    •  & Matthias Frank
  17. Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • James M. Holton
    •  & Stefano Marchesini
  18. Department of Chemistry, Biochemistry and Biophysics, University of Gothenburg, SE-405 30 Gothenburg, Sweden

    • Richard Neutze
  19. Institut für Optik und Atomare Physik, Technische Universität Berlin, Hardenbergstrasse 36, 10623 Berlin, Germany

    • Sebastian Schorb
    • , Daniela Rupp
    • , Marcus Adolph
    •  & Tais Gorkhover
  20. Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Box 590, S-751 24 Uppsala, Sweden

    • Inger Andersson


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H.N.C. and J.C.H.S. conceived the experiment, which was designed with P.F., A.B., R.A.K., J.S., D.P.D., U.W., R.B.D., S. Boutet, M.J.B., D.S., I.S., S.M. and J.H. The CAMP instrument was the responsibility of S.W.E., R.H., D. Rolles, A. Rudenko, C.S., L.F., N.K., P.H., B.R., B.E., A.H., Ch.R., D.P., G.W., L.S., G.H., H. Gorke, J.U., I.S., S.H., G.S., F.S., H.S., K.-U.K., R.A., C.-D.S., F.K., M. Bott, S. Schorb, D. Rupp, M.A., T.G., H.H., L.G., G.P., H. Graafsma and B.N., who designed and set up the instrument and/or developed and operated the pnCCD detectors. C.B., J.D.B. and M.M. set up and aligned the beamline. P.F., M.S.H. and I.G. prepared samples; R.B.D., D.P.D., U.W., J.C.H.S., P.F., L.L. and R.L.S. developed and operated the sample delivery system; H.N.C., A.B., A.A., J.S., D.P.D., U.W., R.B.D., S. Bajt, M.J.B., L.G., J.H., M.M.S., N.T., J.A., S. Stern and J.C.H.S. developed diffraction instrumentation; and M. Barthelmess, M.L., A.B. and K.N. designed and/or fabricated calibration samples. J.K., S.P.H.-R., A.B., H.N.C., J.S. and A.V.M. characterized the focus. H.N.C., J.C.H.S., P.F., A.B., T.A.W., R.A.K., A.A., J.S., D.P.D., U.W., R.B.D., I.S., N.C., R.L.S., M.S.H., L.L., M. Bott, S.W.E., R.H., D. Rolles, A. Rudenko, M.L., C.B., J.U., L.F., J.D.B., M.M., M.F., C.Y.H., R.G.S., G.J.W., A. Rocker, M.S., O.J., I.A. and J.H. carried out the experiment. A.B., T.A.W., R.A.K., A.A., F.R.N.C.M., A.V.M., L.L., T.R.M.B., N.C., L.F., N.K., R.N., G.W., P.H., C.C., J.M.H., I.S., J.H., H.N.C. and J.C.H.S. analysed the data. A.V.M. performed the Bragg shape phase retrieval. T.A.W. and R.A.K. merged the 3D data. R.F. collected and evaluated the reference data set; R.A.K., T.A.W., J.M.H. and R.F. refined the structure and calculated the electron density maps; and H.N.C., P.F., J.C.H.S. and I.S. wrote the manuscript with discussion and improvements from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Henry N. Chapman.

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    Supplementary Information

    The file contains Supplementary Table 1, Supplementary Figures 1-4 with legends, Supplementary Methods and Data and additional references.

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