Protocol | Published:

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

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    & Biocrystallography: past, present, future. HFSP J. 4, 109–121 (2010).

  2. 2.

    , & How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006).

  3. 3.

    , & Structurally enabled discovery of adenosine A2A receptor antagonists. Chem. Rev. 117, 21–37 (2017).

  4. 4.

    Developments in X-ray crystallographic structure determination of biological macromolecules. Science 343, 1102–1108 (2014).

  5. 5.

    High-throughput crystallography for structural genomics. Curr. Opin. Struct. Biol. 19, 573–584 (2009).

  6. 6.

    et al. High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline. Acta Crystallogr. D Biol. Crystallogr. 62, 1162–1169 (2006).

  7. 7.

    , & Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination. J. Struct. Biol. 142, 218–231 (2003).

  8. 8.

    et al. SLS crystallization platform at beamline X06DA—a fully automated pipeline enabling in situ X-ray diffraction screening. Cryst. Growth Des. 11, 916–923 (2011).

  9. 9.

    et al. In-plate protein crystallization, in situ ligand soaking and X-ray diffraction. Acta Crystallogr. D Biol. Crystallogr. 67, 747–755 (2011).

  10. 10.

    et al. Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography. Acta Crystallogr. D Biol. Crystallogr. 71, 1777–1787 (2015).

  11. 11.

    & Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. USA 93, 14532–14535 (1996).

  12. 12.

    A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes. Acta Crystallogr. F Struct. Biol. Commun. 71, 3–18 (2015).

  13. 13.

    & Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).

  14. 14.

    et al. Application of in situ diffraction in high-throughput structure determination platforms. Methods Mol. Biol. 1261, 233–253 (2015).

  15. 15.

    et al. In situ macromolecular crystallography using microbeams. Acta Crystallogr. D Biol. Crystallogr. 68, 592–600 (2012).

  16. 16.

    , , & Applications of thin-film sandwich crystallization platforms. Acta Crystallogr. F Struct. Biol. Commun. 72, 313–319 (2016).

  17. 17.

    et al. CrystalDirect: a new method for automated crystal harvesting based on laser-induced photoablation of thin films. Acta Crystallogr. D Biol. Crystallogr. 68, 1393–1399 (2012).

  18. 18.

    et al. In meso in situ serial X-ray crystallography of soluble and membrane proteins. Acta Crystallogr. D Biol. Crystallogr. 71, 1238–1256 (2015).

  19. 19.

    et al. In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures. Acta Crystallogr. D Biol. Crystallogr. 72, 93–112 (2016).

  20. 20.

    et al. ChipX: a novel microfluidic chip for counter-diffusion crystallization of biomolecules and in situ crystal analysis at room temperature. Cryst. Growth Des. 13, 3333–3340 (2013).

  21. 21.

    et al. Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction. IUCrJ 1, 349–360 (2014).

  22. 22.

    et al. In situ serial Laue diffraction on a microfluidic crystallization device. J. Appl. Crystallogr. 47, 1975–1982 (2014).

  23. 23.

    et al. Chaotic mixer for microchannels. Science 295, 647–651 (2002).

  24. 24.

    et al. A plug-based microfluidic system for dispensing lipidic cubic phase (LCP) material validated by crystallizing membrane proteins in lipidic mesophases. Microfluid. Nanofluidics 8, 789–798 (2010).

  25. 25.

    , , , & X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination. Cryst. Growth Des. 14, 4886–4890 (2014).

  26. 26.

    et al. X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization. Biomicrofluidics 11, 024118-1–024118-13 (2017).

  27. 27.

    et al. In situ data collection and structure refinement from microcapillary protein crystallization. J. Appl. Crystallogr. 38, 900–905 (2005).

  28. 28.

    et al. In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA. Acta Crystallogr. F Struct. Biol. Commun. 68, 1307–1310 (2012).

  29. 29.

    et al. X-ray diffraction of protein crystal grown in a nano-liter scale droplet in a microchannel and evaluation of its applicability. Anal. Sci. 28, 65–68 (2012).

  30. 30.

    et al. Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography. Struct. Dyn. 2, 054302 (2015).

  31. 31.

    et al. Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography. Acta Crystallogr. D Biol. Crystallogr. 72, 944–955 (2012).

  32. 32.

    et al. Chrystallography on a chip. Acta Crystallogr. D Biol. Crystallogr. 68, 321–323 (2012).

  33. 33.

    et al. A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering. Sci. Rep. 5 10451 (2015).

  34. 34.

    & Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins. J. Appl. Crystallogr. 36, 1372–1377 (2003).

  35. 35.

    Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin. Struct. Biol. 21, 559–566 (2011).

  36. 36.

    et al. A versatile system for high-throughput in situ X-ray screening and data collection of soluble and membrane-protein crystals. Cryst. Growth Des. 16, 6318–6326 (2016).

  37. 37.

    , & Guiding principles of specimen preservation for confocal fluorescence microscopy. in Handbook of Biological Confocal Microscopy. 3rd edn (ed Pawley, J. B.) Ch. 18 (Springer, 2006).

  38. 38.

    et al. One-micron beams for macromolecular crystallography at GM/CA-CAT. AIP Conf. Proc. 1234, 419–422 (2010).

  39. 39.

    et al. The PILATUS 1M detector. J. Synchrotron Rad. 13, 120–130 (2006).

  40. 40.

    et al. EIGER: next generation single photon counting detector for X-ray applications. Nucl. Instrum. Methods Phys. Res. A 650, 79–83 (2011).

  41. 41.

    et al. Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 μm size X-ray synchrotron beam. J. R. Soc. Interface 6, S587–S597 (2009).

  42. 42.

    et al. Tightly integrated single- and multi-crystal data collection strategy calculation and parallelized data processing in JBluIce beamline control system. J. Appl. Crystallogr. 47, 1992–1999 (2014).

  43. 43.

    et al. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr. Sect. D: Biol. Crystallogr. 69, 1617–1632 (2013).

  44. 44.

    & Crystallization of soluble proteins in vapor diffusion for X-ray crystallography. Nat. Protoc. 2, 1633–1651 (2007).

  45. 45.

    & Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization. Biophys. J. 85, 3084–3096 (2003).

  46. 46.

    , & Crystallogenesis of membrane proteins mediated by polymer-bounded lipid nanodiscs. Structure 25, 384–392 (2017).

  47. 47.

    Macromolecular cryocrystallography—methods for cooling and mounting protein crystals at cryogenic temperatures. Methods 34, 415–423 (2004).

  48. 48.

    , , , & History and perspectives of A2A adenosine receptor antagonists as potential therapeutic agents. Med. Res. Rev. 35, 790–848 (2015).

  49. 49.

    et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012).

  50. 50.

    XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  51. 51.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

  52. 52.

    et al. Collaboration gets the most out of software. eLIFE 2, e01456 (2013).

  53. 53.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  54. 54.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  55. 55.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  56. 56.

    G protein-coupled receptor Rhodopsin. Annu. Rev. Biochem. 75, 743–767 (2006).

  57. 57.

    , , , & Crystal structure of the ligand-free G-protein-coupled receptor Opsin. Nature 454, 183–188 (2008).

  58. 58.

    , , & Diffusible ligand all-trans-retinal activates Opsin via a palmitoylation-dependent mechanism. J. Biol. Chem. 275, 6189–6194 (2000).

  59. 59.

    & How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

  60. 60.

    et al. Opsin, a structural model for olfactory receptors? Angew. Chem. Int. Ed. 52, 11021–11024 (2013).

  61. 61.

    et al. Acoustically mounted microcrystals yield high-resolution X-ray structures. Biochemistry 50, 4399–4401 (2011).

Download references

Acknowledgements

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.

Affiliations

  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

Authors

  1. Search for Jana Broecker in:

  2. Search for Takefumi Morizumi in:

  3. Search for Wei-Lin Ou in:

  4. Search for Viviane Klingel in:

  5. Search for Anling Kuo in:

  6. Search for David J Kissick in:

  7. Search for Andrii Ishchenko in:

  8. Search for Ming-Yue Lee in:

  9. Search for Shenglan Xu in:

  10. Search for Oleg Makarov in:

  11. Search for Vadim Cherezov in:

  12. Search for Craig M Ogata in:

  13. Search for Oliver P Ernst in:

Contributions

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.

Videos

  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).

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2017.135

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.