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High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions

Nature Protocols volume 13, pages 260292 (2018) | Download Citation


Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.

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We thank members of the Ernst group, in particular A.R. Balo and Y. Shen, for their contributions to the original work. We acknowledge the MADLab and the Gerstein Library, mainly E. Lenton and M. Spears (University of Toronto), for admission to the 3D-printing facility. We also thank A. Trnka (Saunders) for supplying us with various spacers. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We specifically thank the staff at the GM/CA beamline. Videos of the Irelec CATS robot mounting 1-well holders were shot in the LS-CAT ID-D endstation, Sector 21, at the APS, for which we acknowledge J. Brunzelle, a member of the LS-CAT staff. S. Haider (Formulatrix) kindly calibrated a Mylar in situ plate for use with the Rock Imager. For fruitful discussions and carefully reading the manuscript, we are grateful to E.F. Pai (University of Toronto) and S. Keller (University of Kaiserslautern). This work was supported by a Research Fellowship from the German Research Foundation (DFG) to J.B. (BR 5124/1-1), by National Institutes of Health grant R01 GM108635 (to V.C.), and by funding from the Canadian Institute for Advanced Research (CIFAR; to O.P.E.). O.P.E. holds a Canada Excellence Research Chair Award and the Anne and Max Tanenbaum Chair in Neuroscience at the University of Toronto.

Author information

Author notes

    • Jana Broecker

    Present address: Heptares Therapeutics Ltd., Welwyn Garden City, UK.


  1. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

    • Jana Broecker
    • , Takefumi Morizumi
    • , Wei-Lin Ou
    • , Viviane Klingel
    • , Anling Kuo
    •  & Oliver P Ernst
  2. GM/CA at Advanced Photon Source, Argonne National Laboratory, Lemont, Illinois, USA.

    • David J Kissick
    • , Shenglan Xu
    • , Oleg Makarov
    •  & Craig M Ogata
  3. Department of Chemistry, The Bridge Institute, University of Southern California, Los Angeles, Los Angeles, California, USA.

    • Andrii Ishchenko
    • , Ming-Yue Lee
    •  & Vadim Cherezov
  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

    • Oliver P Ernst


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J.B. and O.P.E. designed the research. J.B. wrote the manuscript. All authors commented on the manuscript. V.C. supervised A2A work. J.B., T.M., W.-L.O., A.I., and M.-Y.L. grew crystals, and collected and analyzed the data. V.K. helped with holder designs. A.K. grew SWMb crystals. D.J.K. and C.M.O. helped with data collection and implementation of the Mylar in situ setup at the APS synchrotron. S.X. and O.M. designed the GM/CA adaptors, including the translation stages.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jana Broecker or Oliver P Ernst.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5, Supplementary Tables 1 and 2, the Supplementary Methods, and Supplementary Manuals 1 and 2.

Zip files

  1. 1.

    Supplementary Data 1–4

    Supplementary Data 1. Printer files for 1-well G-holders.  Supplementary Data 2. Printer files for 4-well G-holders.  Supplementary Data 3. Printer files for GT-holders.  Supplementary Data 4. Printer files for GAT-holders.


  1. 1.

    GM/CA robot. Utomounter robot at GM/CA mounting and demounting 1-well holders stored in liquid nitrogen.

  2. 2.

    Irelec CATS robot. Commercially available Irelec CATS robot mounting and demounting 1-well holders stored in sample vials in liquid nitrogen.

  3. 3.

    Tweezer holder. Loading an individual well to a tweezer holder (1-well G-holder).

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