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In vivo protein crystallization opens new routes in structural biology

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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Figure 1: Light microscopic and EM analysis of Sf9 insect cells with embedded in vivo crystals.
Figure 2: Serial femtosecond crystallography of in vivo TbCatB crystals.
Figure 3: Structure of solubilized and recrystallized TbCatB solved by conventional X-ray crystallography.

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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.

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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.

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Correspondence to Michael Duszenko.

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The authors declare no competing financial interests.

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Supplementary Figures 1–3, Supplementary Tables 1–3 and Supplementary Note (PDF 1819 kb)

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Koopmann, R., Cupelli, K., Redecke, L. et al. In vivo protein crystallization opens new routes in structural biology. Nat Methods 9, 259–262 (2012). https://doi.org/10.1038/nmeth.1859

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