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The collection of MicroED data for macromolecular crystallography

Nature Protocols volume 11, pages 895–904 (2016)Cite this article

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Abstract

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

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Figure 1: Structures determined by MicroED.
Figure 2: Flow diagram for MicroED data collection protocol.
Figure 3: Representative protein microcrystal images.
Figure 4: Assembled Vitrobot coolant container.
Figure 5: Setting the eucentric height correctly.
Figure 6: Low-dose settings menu on the FEI Tecnai F20 microscope.
Figure 7: Example images from search, focus and exposure modes from a model MicroED sample.

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References

  1. Chapman, H.N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011).

    Article  CAS  Google Scholar 

  2. Moukhametzianov, R. et al. Protein crystallography with a micrometre-sized synchrotron-radiation beam. Acta Crystallogr. D Biol. Crystallogr. 64, 158–166 (2008).

    Article  CAS  Google Scholar 

  3. Nannenga, B.L., Shi, D., Leslie, A.G. & Gonen, T. High-resolution structure determination by continuous-rotation data collection in MicroED. Nat. Methods 11, 927–930 (2014).

    Article  CAS  Google Scholar 

  4. Shi, D., Nannenga, B.L., Iadanza, M.G. & Gonen, T. Three-dimensional electron crystallography of protein microcrystals. Elife 2, e01345 (2013).

    Article  Google Scholar 

  5. Zhang, Y.B. et al. Single-crystal structure of a covalent organic framework. J. Am. Chem. Soc. 135, 16336–16339 (2013).

    Article  CAS  Google Scholar 

  6. Wan, W., Sun, J.L., Su, J., Hovmoller, S. & Zou, X.D. Three-dimensional rotation electron diffraction: software RED for automated data collection and data processing. J. Appl. Crystallogr. 46, 1863–1873 (2013).

    Article  CAS  Google Scholar 

  7. Mugnaioli, E., Gorelik, T. & Kolb, U. “Ab initio” structure solution from electron diffraction data obtained by a combination of automated diffraction tomography and precession technique. Ultramicroscopy 109, 758–765 (2009).

    Article  CAS  Google Scholar 

  8. Jiang, J.X. et al. Synthesis and structure determination of the hierarchical meso-microporous zeolite ITQ-43. Science 333, 1131–1134 (2011).

    Article  CAS  Google Scholar 

  9. Gemmi, M., La Placa, M.G.I., Galanis, A.S., Rauch, E.F. & Nicolopoulos, S. Fast electron diffraction tomography. J. Appl. Crystallogr. 48, 718–727 (2015).

    Article  CAS  Google Scholar 

  10. Nannenga, B.L., Shi, D., Hattne, J., Reyes, F.E. & Gonen, T. Structure of catalase determined by MicroED. Elife 3, e03600 (2014).

    Article  Google Scholar 

  11. Rodriguez, J.A. et al. Structure of the toxic core of alpha-synuclein from invisible crystals. Nature 525, 486–490 (2015).

    Article  CAS  Google Scholar 

  12. Yonekura, K., Kato, K., Ogasawara, M., Tomita, M. & Toyoshima, C. Electron crystallography of ultrathin 3D protein crystals: atomic model with charges. Proc. Natl. Acad. Sci. USA 112, 3368–3373 (2015).

    Article  CAS  Google Scholar 

  13. Liu, W. et al. Serial femtosecond crystallography of G protein-coupled receptors. Science 342, 1521–1524 (2013).

    Article  CAS  Google Scholar 

  14. Sugahara, M. et al. Grease matrix as a versatile carrier of proteins for serial crystallography. Nat. Methods 12, 61–63 (2015).

    Article  CAS  Google Scholar 

  15. Weierstall, U., Spence, J.C. & Doak, R.B. Injector for scattering measurements on fully solvated biospecies. Rev. Sci. Instrum. 83, 035108 (2012).

    Article  CAS  Google Scholar 

  16. Gonen, T. The collection of high-resolution electron diffraction data. Methods Mol. Biol. 955, 153–169 (2013).

    Article  CAS  Google Scholar 

  17. Battye, T.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  Google Scholar 

  18. Leslie, A.G.W. & Powell, H.R. Processing diffraction data with MOSFLM. NATO Sci. Ser. 245, 41–51 (2007).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  21. Waterman, D.G. et al. The DIALS framework for integration software. CCP4 Newslett. Protein Crystallogr. 49, 16–19 (2013).

    Google Scholar 

  22. Brunger, A.T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  Google Scholar 

  23. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D Biol. Crystallogr. 60, 2210–2221 (2004).

    Article  CAS  Google Scholar 

  26. Sheldrick, G.M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 479–485 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Huang, P.S. et al. High thermodynamic stability of parametrically designed helical bundles. Science 346, 481–485 (2014).

    Article  CAS  Google Scholar 

  29. Hattne, J. et al. MicroED data collection and processing. Acta. Crystallogr. A Found. Adv. 71, 353–360 (2015).

    Article  CAS  Google Scholar 

  30. Nannenga, B.L., Iadanza, M.G., Vollmar, B.S. & Gonen, T. Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy. Curr. Protoc. Protein Sci. Chapter 17, Unit17.15 (2013).

    PubMed  Google Scholar 

  31. Wampler, R.D. et al. Selective detection of protein crystals by second harmonic microscopy. J. Am. Chem. Soc. 130, 14076–14077 (2008).

    Article  CAS  Google Scholar 

  32. Stevenson, H.P. et al. Use of transmission electron microscopy to identify nanocrystals of challenging protein targets. Proc. Natl. Acad. Sci. USA 111, 8470–8475 (2014).

    Article  CAS  Google Scholar 

  33. Grassucci, R.A., Taylor, D. & Frank, J. Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat. Protoc. 3, 330–339 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

Work in the Gonen lab is supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Dan Shi, Brent L Nannenga, M Jason de la Cruz, Jinyang Liu, Steven Sawtelle, Francis E Reyes, Johan Hattne & Tamir Gonen

  2. Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Guillermo Calero

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Contributions

D.S., B.L.N. and T.G. wrote the manuscript. D.S., B.L.N., M.J.d.l.C., F.E.R., J.H. and T.G. designed and performed the experiments, and edited the manuscript. J.L. and J.H. worked on software design. D.S., T.G. and S.S. designed and built the rotation controller. G.C. provided Fig. 2d,h. All authors read and approved the manuscript.

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Correspondence to Tamir Gonen.

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Shi, D., Nannenga, B., de la Cruz, M. et al. The collection of MicroED data for macromolecular crystallography. Nat Protoc 11, 895–904 (2016). https://doi.org/10.1038/nprot.2016.046

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