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.
The file contains Supplementary Table 1, Supplementary Figures 1-4 with legends, Supplementary Methods and Data and additional references.
About this article
Nature Communications (2019)