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The cryo-EM method microcrystal electron diffraction (MicroED)


In 2013 we established a cryo-electron microscopy (cryo-EM) technique called microcrystal electron diffraction (MicroED). Since that time, data collection and analysis schemes have been fine-tuned, and structures for more than 40 different proteins, oligopeptides and organic molecules have been determined. Here we review the MicroED technique and place it in context with other structure-determination methods. We showcase example structures solved by MicroED and provide practical advice to prospective users.

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  1. 1.

    Cheng, Y., Grigorieff, N., Penczek, P. A. & Walz, T. A primer to single-particle cryo-electron microscopy. Cell 161, 438–449 (2015).

  2. 2.

    Beck, M. & Baumeister, W. Cryo-electron tomography: can it reveal the molecular sociology of cells in atomic detail? Trends Cell Biol. 26, 825–837 (2016).

  3. 3.

    Wisedchaisri, G., Reichow, S. L. & Gonen, T. Advances in structural and functional analysis of membrane proteins by electron crystallography. Structure 19, 1381–1393 (2011).

  4. 4.

    Glaeser, R. M. Review: electron crystallography: present excitement, a nod to the past, anticipating the future. J. Struct. Biol. 128, 3–14 (1999).

  5. 5.

    Nannenga, B. L. & Gonen, T. MicroED opens a new era for biological structure determination. Curr. Opin. Struct. Biol. 40, 128–135 (2016).

  6. 6.

    Rodriguez, J. A., Eisenberg, D. S. & Gonen, T. Taking the measure of MicroED. Curr. Opin. Struct. Biol. 46, 79–86 (2017).

  7. 7.

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

  8. 8.

    Nannenga, B. L., Shi, D., Leslie, A. G. W. & Gonen, T. High-resolution structure determination by continuous-rotation data collection in MicroED. Nat. Methods 11, 927–930 (2014). This work introduced the ‘continuous rotation’ method of MicroED data collection, which is currently the standard procedure for data collection.

  9. 9.

    Gonen, T., Sliz, P., Kistler, J., Cheng, Y. & Walz, T. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429, 193–197 (2004).

  10. 10.

    Gonen, T. et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438, 633–638 (2005).

  11. 11.

    Henderson, R. & Unwin, P. N. Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257, 28–32 (1975).

  12. 12.

    Henderson, R. et al. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 213, 899–929 (1990).

  13. 13.

    Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M. & Henderson, R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259, 393–421 (1996).

  14. 14.

    Subramaniam, S. & Henderson, R. Crystallographic analysis of protein conformational changes in the bacteriorhodopsin photocycle. Biochim. Biophys. Acta 1460, 157–165 (2000).

  15. 15.

    Kühlbrandt, W., Wang, D. N. & Fujiyoshi, Y. Atomic model of plant light-harvesting complex by electron crystallography. Nature 367, 614–621 (1994).

  16. 16.

    Unwin, P. N. & Henderson, R. Molecular structure determination by electron microscopy of unstained crystalline specimens. J. Mol. Biol. 94, 425–440 (1975).

  17. 17.

    Dorset, D. L. & Parsons, D. F. Electron-diffraction from single, fully-hydrated, ox liver catalase microcrystals. Acta Crystallogr. A 31, 210–215 (1975).

  18. 18.

    Dorset, D. L. & Parsons, D. F. Thickness measurements of wet protein crystals in electron-microscope. J. Appl. Crystallogr. 8, 12–14 (1975).

  19. 19.

    Taylor, K. A. & Glaeser, R. M. Electron microscopy of frozen hydrated biological specimens. J. Ultrastruct. Res. 55, 448–456 (1976).

  20. 20.

    Jiang, L., Georgieva, D., Zandbergen, H. W. & Abrahams, J. P. Unit-cell determination from randomly oriented electron-diffraction patterns. Acta Crystallogr. D Biol. Crystallogr. 65, 625–632 (2009).

  21. 21.

    Nederlof, I., van Genderen, E., Li, Y. W. & Abrahams, J. P. A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals. Acta Crystallogr. D Biol. Crystallogr. 69, 1223–1230 (2013).

  22. 22.

    Iadanza, M. G. & Gonen, T. A suite of software for processing MicroED data of extremely small protein crystals. J. Appl. Crystallogr. 47, 1140–1145 (2014).

  23. 23.

    Hattne, J., Shi, D., de la Cruz, M. J., Reyes, F. E. & Gonen, T. Modeling truncated pixel values of faint reflections in MicroED images. J. Appl. Crystallogr. 49, 1029–1034 (2016).

  24. 24.

    de la Cruz, M. J. et al. Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED. Nat. Methods 14, 399–402 (2017). Here several methods for the fragmentation of larger, and in some cases poorly ordered, crystals into microcrystals suitable for MicroED are described.

  25. 25.

    Luo, F. et al. Atomic structures of FUS LC domain segments reveal bases for reversible amyloid fibril formation. Nat. Struct. Mol. Biol. 25, 341–346 (2018).

  26. 26.

    Duyvesteyn, H. M. E. et al. Machining protein microcrystals for structure determination by electron diffraction. Proc. Natl Acad. Sci. USA 115, 9569–9573 (2018).

  27. 27.

    Kolb, U., Mugnaioli, E. & Gorelik, T. E. Automated electron diffraction tomography—a new tool for nano crystal structure analysis. Cryst. Res. Technol. 46, 542–554 (2011).

  28. 28.

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

  29. 29.

    Mugnaioli, E. et al. Ab initio structure determination of vaterite by automated electron diffraction. Angew. Chem. Int. Ed. Engl. 51, 7041–7045 (2012).

  30. 30.

    Feyand, M. et al. Automated diffraction tomography for the structure elucidation of twinned, sub-micrometer crystals of a highly porous, catalytically active bismuth metal-organic framework. Angew. Chem. Int. Ed. Engl. 51, 10373–10376 (2012).

  31. 31.

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

  32. 32.

    Gorelik, T. E., Stewart, A. A. & Kolb, U. Structure solution with automated electron diffraction tomography data: different instrumental approaches. J. Microsc. 244, 325–331 (2011).

  33. 33.

    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).

  34. 34.

    Simancas, J. et al. Ultrafast electron diffraction tomography for structure determination of the new zeolite ITQ-58. J. Am. Chem. Soc. 138, 10116–10119 (2016).

  35. 35.

    Zhang, C. et al. An extra-large-pore zeolite with 24×8×8-ring channels using a structure-directing agent derived from traditional Chinese medicine. Angew. Chem. Int. Ed. Engl. 57, 6486–6490 (2018).

  36. 36.

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

  37. 37.

    Shi, D. et al. The collection of MicroED data for macromolecular crystallography. Nat. Protoc. 11, 895–904 (2016). Detailed protocols for the collection of MicroED data. New users are encouraged to read these protocols prior to using MicroED.

  38. 38.

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

  39. 39.

    Gallagher-Jones, M. et al. Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp. Nat. Struct. Mol. Biol. 25, 131–134 (2018).

  40. 40.

    Jones, C. G. et al. The cryoEM method MicroED as a powerful tool for small molecule structure determination. ACS Cent. Sci. 4, 1587–1592 (2018).

  41. 41.

    Vergara, S. et al. MicroED structure of Au146(p-MBA)57 at subatomic resolution reveals a twinned FCC cluster. J. Phys. Chem. Lett. 8, 5523–5530 (2017).

  42. 42.

    Fromme, P. & Spence, J. C. Femtosecond nanocrystallography using X-ray lasers for membrane protein structure determination. Curr. Opin. Struct. Biol. 21, 509–516 (2011).

  43. 43.

    Smith, J. L., Fischetti, R. F. & Yamamoto, M. Micro-crystallography comes of age. Curr. Opin. Struct. Biol. 22, 602–612 (2012).

  44. 44.

    Yamamoto, M. et al. Protein microcrystallography using synchrotron radiation. IUCrJ 4, 529–539 (2017).

  45. 45.

    Martin-Garcia, J. M., Conrad, C. E., Coe, J., Roy-Chowdhury, S. & Fromme, P. Serial femtosecond crystallography: a revolution in structural biology. Arch. Biochem. Biophys. 602, 32–47 (2016).

  46. 46.

    Spence, J. C. H. X-ray lasers for structure and dynamics in biology. IUCrJ 5, 236–237 (2018).

  47. 47.

    Henderson, R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171–193 (1995).

  48. 48.

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

  49. 49.

    Sawaya, M. R. et al. Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED. Proc. Natl Acad. Sci. USA 113, 11232–11236 (2016). The first structure determined by MicroED that was phased experimentally. In this study, the high-resolution and MicroED data quality allowed structure solution by direct methods.

  50. 50.

    Krotee, P. et al. Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity. eLife 6, e19273 (2017).

  51. 51.

    Seidler, P. M. et al. Structure-based inhibitors of tau aggregation. Nat. Chem. 10, 170–176 (2018).

  52. 52.

    Hughes, M. P. et al. Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks. Science 359, 698–701 (2018).

  53. 53.

    Kissick, D. J., Wanapun, D. & Simpson, G. J. Second-order nonlinear optical imaging of chiral crystals. Annu. Rev. Anal. Chem. (Palo Alto Calif.) 4, 419–437 (2011).

  54. 54.

    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).

  55. 55.

    Barnes, C. O. et al. Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography. Arch. Biochem. Biophys. 602, 61–68 (2016).

  56. 56.

    Stevenson, H. P. et al. Transmission electron microscopy for the evaluation and optimization of crystal growth. Acta Crystallogr. D Struct. Biol. 72, 603–615 (2016).

  57. 57.

    Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J. & Gonen, T. Collection of continuous rotation MicroED data from ion beam-milled crystals of any size. Structure 27, 545–548 (2019).

  58. 58.

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

  59. 59.

    Leslie, A. G. W. & Powell, H. R. Processing diffraction data with mosflm. In Evolving Methods for Macromolecular Crystallography (eds. Read, R. J. & Sussman, J. L.) 41–51 (Springer, 2007).

  60. 60.

    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).

  61. 61.

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

  62. 62.

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

  63. 63.

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

  64. 64.

    Clabbers, M. T. B., Gruene, T., Parkhurst, J. M., Abrahams, J. P. & Waterman, D. G. Electron diffraction data processing with DIALS. Acta Crystallogr. D Struct. Biol. 74, 506–518 (2018).

  65. 65.

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

  66. 66.

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

  67. 67.

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

  68. 68.

    Balbirnie, M., Grothe, R. & Eisenberg, D. S. An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid. Proc. Natl Acad. Sci. USA 98, 2375–2380 (2001).

  69. 69.

    Nelson, R. et al. Structure of the cross-beta spine of amyloid-like fibrils. Nature 435, 773–778 (2005).

  70. 70.

    Sawaya, M. R. et al. Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447, 453–457 (2007).

  71. 71.

    Rodriguez, J. A. et al. Structure of the toxic core of α-synuclein from invisible crystals. Nature 525, 486–490 (2015). First novel structure determined by MicroED. This work paved the way for the many new peptide structures from amyloidogenic proteins.

  72. 72.

    Guenther, E. L. et al. Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation. Nat. Struct. Mol. Biol. 25, 463–471 (2018).

  73. 73.

    Krotee, P. et al. Common fibrillar spines of amyloid-β and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors. J. Biol. Chem. 293, 2888–2902 (2018).

  74. 74.

    Purdy, M. D. et al. MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat. Proc. Natl Acad. Sci. USA 115, 13258–13263 (2018).

  75. 75.

    Wagner, J. M. et al. Crystal structure of an HIV assembly and maturation switch. eLife 5, e17063 (2016).

  76. 76.

    Schmidt, M. Mix and inject: reaction initiation by diffusion for time-resolved macromolecular crystallography. Adv. Condens. Matter Phys. 2013, 167276 (2013).

  77. 77.

    Unwin, N. & Fujiyoshi, Y. Gating movement of acetylcholine receptor caught by plunge-freezing. J. Mol. Biol. 422, 617–634 (2012).

  78. 78.

    Berriman, J. & Unwin, N. Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56, 241–252 (1994).

  79. 79.

    Unwin, N. Acetylcholine receptor channel imaged in the open state. Nature 373, 37–43 (1995).

  80. 80.

    Nogly, P. et al. Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser. Science 361, eaat0094 (2018).

  81. 81.

    Pande, K. et al. Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352, 725–729 (2016).

  82. 82.

    Wu, J. S. & Spence, J. C. Structure and bonding in alpha-copper phthalocyanine by electron diffraction. Acta Crystallogr. A 59, 495–505 (2003).

  83. 83.

    Hattne, J. et al. Analysis of global and site-specific radiation damage in cryo-EM. Structure 26, 759–766 (2018).

  84. 84.

    Garman, E. F. Radiation damage in macromolecular crystallography: what is it and why should we care? Acta Crystallogr. D Biol. Crystallogr. 66, 339–351 (2010).

  85. 85.

    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. 72, 17.15.1–17.15.11 (2013).

  86. 86.

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

  87. 87.

    Nederlof, I., Li, Y. W., van Heel, M. & Abrahams, J. P. Imaging protein three-dimensional nanocrystals with cryo-EM.Acta Crystallogr. D Biol. Crystallogr. 69, 852–859 (2013).

  88. 88.

    van Genderen, E., Li, Y. W., Nederlof, I. & Abrahams, J. P. Lattice filter for processing image data of three-dimensional protein nanocrystals. Acta Crystallogr. D Struct. Biol. 72, 34–39 (2016).

  89. 89.

    Gorelik, T. E., van de Streek, J., Kilbinger, A. F., Brunklaus, G. & Kolb, U. Ab-initio crystal structure analysis and refinement approaches of oligo p-benzamides based on electron diffraction data. Acta Crystallogr. B 68, 171–181 (2012).

  90. 90.

    Gruene, T. et al. Rapid structure determination of microcrystalline molecular compounds using electron diffraction. Angew. Chem. Int. Ed. Engl. 57, 16313–16317 (2018).

  91. 91.

    van Genderen, E. et al. Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector. Acta Crystallogr. A Found. Adv. 72, 236–242 (2016).

  92. 92.

    Clabbers, M. T. B. et al. Protein structure determination by electron diffraction using a single three-dimensional nanocrystal. Acta Crystallogr. D Struct. Biol. 73, 738–748 (2017).

  93. 93.

    de la Cruz, M. J., Martynowycz, M. W., Hattne, J. & Gonen, T. MicroED data collection with SerialEM. Ultramicroscopy 201, 77–80 (2019).

  94. 94.

    Fernández-Busnadiego, R. et al. Insights into the molecular organization of the neuron by cryo-electron tomography. J. Electron Microsc. (Tokyo) 60, S137–S148 (2011).

  95. 95.

    Bartesaghi, A. et al. 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Science 348, 1147–1151 (2015).

  96. 96.

    Liu, S. & Gonen, T. MicroED structure of the NaK ion channel reveals a Na+ partition process into the selectivity filter. Commun. Biol. 1, 38 (2018). The first membrane protein determined by continuous rotation MicroED.

  97. 97.

    Subramanian, G., Basu, S., Liu, H., Zuo, J. M. & Spence, J. C. H. Solving protein nanocrystals by cryo-EM diffraction: multiple scattering artifacts. Ultramicroscopy 148, 87–93 (2015).

  98. 98.

    Vincent, R. & Midgley, P. A. Double conical beam-rocking system for measurement of integrated electron diffraction intensities. Ultramicroscopy 53, 271–282 (1994).

  99. 99.

    Midgley, P. A. & Eggeman, A. S. Precession electron diffraction—a topical review. IUCrJ 2, 126–136 (2015).

  100. 100.

    Gjonnes, J. et al. Structure model for the phase AlmFe derived from three-dimensional electron diffraction intensity data collected by a precession technique. Comparison with convergent-beam diffraction. Acta Crystallogr. A 54, 306–319 (1998).

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We thank all of our collaborators and trainees who have contributed either directly or indirectly to the development of MicroED. We thank G. Calero (University of Pittsburgh) for providing Figs. 4c and 5b. The Gonen laboratory is supported by funding from the Howard Hughes Medical Institute (HHMI). The Nannenga laboratory is supported by the US National Institutes of Health (R01GM124152). MicroED was developed at the Janelia Research Campus of HHMI using HHMI funds and Janelia Visitor Program funds.

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Correspondence to Brent L. Nannenga or Tamir Gonen.

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Fig. 1: Methods in the field of cryo-EM.
Fig. 2: MicroED overview.
Fig. 3: Improvements in MicroED data quality.
Fig. 4: Examples of novel structures determined by MicroED.
Fig. 5: Crystal identification and sample preparation for MicroED.
Fig. 6: Cryo-FIB milling of a thick crystal.
Fig. 7: Comparison of proteinase K data collected with and without an energy filter.
Fig. 8: Dynamics probed in response to radiation damage.