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High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly

  • Louie D. Henderson
  • Morgan Beeby
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)

Abstract

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.

Key words

Electron cryo-tomography Subtomogram averaging Molecular machines Protein self-assembly Structural biology 
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Notes

Acknowledgment

LH was supported by a Biotechnology and Biological Sciences Research Council postgraduate training award and Biotechnology and Biological Sciences Research Council Grant BB/L023091/1 to MB.

References

  1. 1.
    Asano S, Engel BD, Baumeister W (2016) In situ cryo-electron tomography: a post-reductionist approach to structural biology. J Mol Biol 428:332–343. https://doi.org/10.1016/j.jmb.2015.09.030 CrossRefPubMedGoogle Scholar
  2. 2.
    Lučić V, Rigort A, Baumeister W (2013) Cryo-electron tomography: the challenge of doing structural biology in situ. J Cell Biol 202:407–419. https://doi.org/10.1083/jcb.201304193 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Briggs JA (2013) Structural biology in situ—the potential of subtomogram averaging. Curr Opin Struct Biol 23:261–267. https://doi.org/10.1016/j.sbi.2013.02.003 CrossRefPubMedGoogle Scholar
  4. 4.
    Beck M, Baumeister W (2016) Cryo-electron tomography: can it reveal the molecular sociology of cells in atomic detail? Trends Cell Biol 26:825–837. https://doi.org/10.1016/j.tcb.2016.08.006 CrossRefPubMedGoogle Scholar
  5. 5.
    Chen S, Beeby M, Murphy GE et al (2011) Structural diversity of bacterial flagellar motors. EMBO J 30:2972–2981. https://doi.org/10.1038/emboj.2011.186 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Beeby M, Ribardo DA, Brennan CA et al (2016) Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold. Proc Natl Acad Sci 113:E1917–E1926. https://doi.org/10.1073/pnas.1518952113 CrossRefPubMedGoogle Scholar
  7. 7.
    Chang Y-W, Rettberg LA, Treuner-Lange A et al (2016) Architecture of the type IVa pilus machine. Science 351:aad2001. https://doi.org/10.1126/science.aad2001 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhao X, Zhang K, Boquoi T et al (2013) Cryoelectron tomography reveals the sequential assembly of bacterial flagella in Borrelia burgdorferi. Proc Natl Acad Sci 110(35):14390–14395. https://doi.org/10.1073/pnas.1308306110 CrossRefPubMedGoogle Scholar
  9. 9.
    Chang J, Liu X, Rochat RH et al (2012) Reconstructing virus structures from nanometer to near-atomic resolutions with cryo-electron microscopy and tomography. Adv Exp Med Biol 726:49–90. https://doi.org/10.1007/978-1-4614-0980-9_4 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Schur FKM, Obr M, Hagen WJH et al (2016) An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science 353:506–508. https://doi.org/10.1126/science.aaf9620 CrossRefPubMedGoogle Scholar
  11. 11.
    Hagen WJH, Wan W, Briggs JAG (2017) Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J Struct Biol 197:191–198. https://doi.org/10.1016/j.jsb.2016.06.007 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kunz M, Frangakis AS (2017) Three-dimensional CTF correction improves the resolution of electron tomograms. J Struct Biol 197:114–122. https://doi.org/10.1016/j.jsb.2016.06.016 CrossRefPubMedGoogle Scholar
  13. 13.
    Williams DB, Carter CB (2009) Transmission electron microscopy: a textbook for materials science. Springer, BerlinCrossRefGoogle Scholar
  14. 14.
    Woldringh CL (1976) Morphological analysis of nuclear separation and cell division during the life cycle of Escherichia coli. J Bacteriol 125:248–257PubMedPubMedCentralGoogle Scholar
  15. 15.
    Farley MM, Hu B, Margolin W, Liu J (2016) Minicells, back in fashion. J Bacteriol 198:1186–1195. https://doi.org/10.1128/JB.00901-15 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Schorb M, Gaechter L, Avinoam O et al (2017) New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography. J Struct Biol 197:83–93. https://doi.org/10.1016/j.jsb.2016.06.020 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Briegel A, Ladinsky MS, Oikonomou C et al (2014) Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling. eLife 3:e02151. https://doi.org/10.7554/eLife.02151 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51. https://doi.org/10.1016/j.jsb.2005.07.007 CrossRefPubMedGoogle Scholar
  19. 19.
    Zheng QS, Braunfeld MB, Sedat JW, Agard DA (2004) An improved strategy for automated electron microscopic tomography. J Struct Biol 147:91–101. https://doi.org/10.1016/j.jsb.2004.02.005 CrossRefPubMedGoogle Scholar
  20. 20.
    Kremer J, Mastronarde D, McIntosh J (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76CrossRefPubMedGoogle Scholar
  21. 21.
    Nicastro D, Schwartz C, Pierson J et al (2006) The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313:944–948. https://doi.org/10.1126/science.1128618 CrossRefPubMedGoogle Scholar
  22. 22.
    Suloway C, Shi J, Cheng A et al (2009) Fully automated, sequential tilt-series acquisition with Leginon. J Struct Biol 167:11–18. https://doi.org/10.1016/j.jsb.2009.03.019 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ding HJ, Oikonomou CM, Jensen GJ (2015) The Caltech tomography database and automatic processing pipeline. J Struct Biol 192:279–286. https://doi.org/10.1016/j.jsb.2015.06.016 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Russo CJ, Passmore LA (2014) Ultrastable gold substrates for electron cryomicroscopy. Science 346:1377–1380. https://doi.org/10.1126/science.1259530 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Tivol WF, Briegel A, Jensen GJ (2008) An improved cryogen for plunge freezing. Microsc Microanal 14:375–379. https://doi.org/10.1017/S1431927608080781 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Jain T, Sheehan P, Crum J et al (2012) Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J Struct Biol 179:68–75. https://doi.org/10.1016/j.jsb.2012.04.020 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Cao M, Takaoka A, Zhang H-B, Nishi R (2011) An automatic method of detecting and tracking fiducial markers for alignment in electron tomography. J Electron Microsc 60:39–46. https://doi.org/10.1093/jmicro/dfq076 CrossRefGoogle Scholar
  28. 28.
    Mastronarde DN, Held SR (2017) Automated tilt series alignment and tomographic reconstruction in IMOD. J Struct Biol 197:102–113. https://doi.org/10.1016/j.jsb.2016.07.011 CrossRefPubMedGoogle Scholar
  29. 29.
    Morado DR, Hu B, Liu J (2016) Using tomoauto – a protocol for high-throughput automated cryo-electron tomography. J Vis Exp:e53608. https://doi.org/10.3791/53608
  30. 30.
    Bharat TAM, Scheres SHW (2016) Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat Protoc 11:2054–2065. https://doi.org/10.1038/nprot.2016.124 CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Life SciencesImperial College of LondonLondonUK

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