Abstract

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

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Acknowledgements

FEL experiments were carried out at LCLS in June 2010 (TbCatB) and in August 2011 (TbIMPDH), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The X-ray diffraction experiments on recrystallized TbCatB crystals were carried out at beamline X06DA of the Swiss Light Source (Villigen, Switzerland). This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG), from the Swedish Research Council, from the Knut och Alice Wallenbergs Stiftelse, from the European Research Council, as well as by US National Science Foundation award MCB-1021557. R.K. received a fellowship from the Landesgraduiertenförderung Baden-Württemberg. L.R., D. Rehders and C. Betzel thank the German Federal Ministry for Education and Research for funding (grants 01KX0806 and 01KX0807). Support from the Hamburg Ministry of Science and Research and Joachim Herz Stiftung as part of the Hamburg Initiative for Excellence in Research and the Hamburg School for Structure and Dynamics in infection, and from the DFG Cluster of Excellence “Inflammation at Interfaces” (EXC 306) is gratefully acknowledged. Funding for the development and operation of the CFEL-ASG multipurpose (CAMP) instrument within the Advanced Study Group at the Center for Free-Electron Laser Science was provided by the Max Planck Society. M.J.B., R.G.S. and C.Y.H. acknowledge funding from the US Department of Energy Office of Basic Energy Sciences through the Photon Ultrafast Laser Science and Engineering (PULSE) Institute at the Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory.

Author information

Author notes

    • Andrew Aquila
    • , Nicola Coppola
    •  & Janos Hajdu

    Present addresses: European XFEL GmbH, Hamburg, Germany (A.A. and N.C.) and Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA (J.H.).

    • Rudolf Koopmann
    • , Karolina Cupelli
    •  & Lars Redecke

    These authors contributed equally to this work.

Affiliations

  1. Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

    • Rudolf Koopmann
    • , Karolina Cupelli
    • , Thilo Stehle
    •  & Michael Duszenko
  2. Joint Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg, and Institute of Biochemistry, University of Lübeck, at Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany.

    • Lars Redecke
    •  & Dirk Rehders
  3. Institute for Experimental Physics, University of Hamburg, Hamburg, Germany.

    • Karol Nass
    •  & Henry N Chapman
  4. Center for Free-Electron Laser Science, DESY, Hamburg, Germany.

    • Daniel P DePonte
    • , Thomas A White
    • , Francesco Stellato
    • , Mengning Liang
    • , Andrew Aquila
    • , Anton Barty
    • , Carl Caleman
    • , Nicola Coppola
    • , Holger Fleckenstein
    • , Lars Gumprecht
    • , Andrew V Martin
    • , Joachim Schulz
    • , Stephan Stern
    •  & Henry N Chapman
  5. Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

    • Jakob Andreasson
    • , Jan Davidsson
    • , Tomas Ekeberg
    • , Janos Hajdu
    • , M Marvin Seibert
    •  & Nicusor Timneanu
  6. Photon Science, DESY, Hamburg, Germany.

    • Sasa Bajt
    • , Miriam Barthelmess
    • , Heinz Graafsma
    • , Helmut Hirsemann
    •  & Cornelia B Wunderer
  7. Photon Ultrafast Laser Science and Engineering (PULSE) Institute, Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory, Menlo Park, California, USA.

    • Michael J Bogan
    • , Christina Y Hampton
    •  & Raymond G Sierra
  8. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA.

    • Christoph Bostedt
    • , Sébastien Boutet
    • , John D Bozek
    • , Marc Messerschmidt
    •  & Garth J Williams
  9. Department of Physics, Arizona State University, Tempe, Arizona, USA.

    • R Bruce Doak
    • , Richard A Kirian
    • , Xiaoyu Wang
    • , Uwe Weierstall
    •  & John C H Spence
  10. Max Planck Advanced Study Group, Center for Free-Electron Laser Science, Hamburg, Germany.

    • Sascha W Epp
    • , Benjamin Erk
    • , Lutz Foucar
    • , Daniel Rolles
    • , Benedikt Rudek
    • , Artem Rudenko
    • , Ilme Schlichting
    • , Lothar Strüder
    •  & Joachim Ullrich
  11. Max-Planck-Institut für Kernphysik, Heidelberg, Germany.

    • Sascha W Epp
    • , Benjamin Erk
    • , Benedikt Rudek
    • , Artem Rudenko
    •  & Joachim Ullrich
  12. Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.

    • Lutz Foucar
    • , Stephan Kassemeyer
    • , Lukas Lomb
    • , Daniel Rolles
    • , Ilme Schlichting
    •  & Robert L Shoeman
  13. PNSensor GmbH, Munich, Germany.

    • Andreas Hartmann
    • , Robert Hartmann
    • , Peter Holl
    • , Christian Reich
    •  & Heike Soltau
  14. Max-Planck-Institut Halbleiterlabor, Munich, Germany.

    • Günter Hauser
    • , Nils Kimmel
    • , Lothar Strüder
    •  & Georg Weidenspointner
  15. Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona, USA.

    • Mark S Hunter
    •  & Petra Fromme
  16. National Energy Research Scientific Computing Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

    • Filipe R N C Maia
  17. Max-Planck-Institut für extraterrestrische Physik, Garching, Germany.

    • Nils Kimmel
    • , Lothar Strüder
    •  & Georg Weidenspointner
  18. University of Siegen, Siegen, Germany.

    • Lothar Strüder
  19. Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

    • Thilo Stehle
  20. Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.

    • M Marvin Seibert
    •  & Christian Betzel

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Contributions

R.K., K.C. and L.R. contributed equally to this work. R.K. performed the in vivo crystallization experiments under the supervision of M.D.; K.C. prepared samples for synchrotron X-ray crystallography and collected and analyzed data under the supervision of T.S.; H.N.C. and J.C.H.S. conceived the SFX experiment, which was designed with P.F., A.B., R.A.K., J.S., D.P.D., U.W., R.B.D., M.J.B., I.S., H.F. and J.H.; FEL samples were prepared by L.R., D. Rehders and C. Betzel; SFX experiments were carried out by L.R., K.N., H.N.C., D.P.D., F.S., M.L., T.A.W., A.A., M.J.B., C.Y.H., R.G.S., U.W., A.B., R.A.K., R.B.D., N.C., R.L.S., L.L., J.D., M.S.H., C. Bostedt, J.D.B., S. Boutet and G.J.W.; beamline setup was done by C. Bostedt, J.D.B., S. Boutet, G.J.W. and M.M. The delivery system was developed and operated by R.B.D., D.P.D., U.W., J.C.H.S., P.F., L.L. and R.L.S.; S.W.E., B.E., L.F., H.G., A.H., R.H., G.H., H.H., P.H., N.K., C.R., D. Rolles, B.R., A.R., H.S., L.S., J.U., C.G.W. and G.W. operated the CAMP instrument and the pn junction charge-coupled devices and developed the software for pnCCD readout. Diffraction instrumentation was developed and calibrated by H.N.C., A.B., A.A., J.S., D.P.D., U.W., R.B.D., M.J.B., L.G., J.H., M.M.S., N.T., J.A., S.S., S. Bajt, M.B. and J.C.H.S. Data were analyzed by T.A.W., K.N., F.S., A.B., R.A.K., A.A., F.R.N.C.M., A.V.M., L.L., N.C., L.F., N.K., G.W., P.H., C.C., I.S., T.E., J.H., S.K., X.W., H.N.C. and J.C.H.S. The manuscript was prepared by L.R., M.D., C. Betzel and T.S. with discussion and improvements from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael Duszenko.

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DOI

https://doi.org/10.1038/nmeth.1859

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