Skip to main content
SpringerLink
Log in

Getting Started with In Situ Cryo-Electron Tomography

  • Protocol
  • First Online:
cryoEM

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2215))

Abstract

Cryo-electron tomography (cryo-ET) is an extremely powerful tool which is used to image cellular features in their close-to-native environment at a resolution where both protein structure and membrane morphology can be revealed. Compared to conventional electron microscopy methods for biology, cryo-ET does not include the use of potentially artifact generating agents for sample fixation or visualization. Despite its obvious advantages, cryo-ET has not been widely adopted by cell biologists. This might originate from the overwhelming and constantly growing number of complex ways to record and process data as well as the numerous methods available for sample preparation. In this chapter, we will take one step back and guide the reader through the essential steps of sample preparation using mammalian cells, as well as the basic steps involved in data recording and processing. The described protocol will allow the reader to obtain data that can be used for morphological analysis and precise measurements of biological structures in their cellular environment. Furthermore, this data can be used for more elaborate structural analysis by applying further image processing steps like subtomogram averaging, which is required to determine the structure of proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Porter KR, Claude A, Fullam EF (1945) A study of tissue culture cells by Electron microscopy. J Exp Med 81:233–246. https://doi.org/10.1084/jem.81.3.233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Palade GE (1952) The fine structure of mitochondria. Anat Rec 114:427–451. https://doi.org/10.1002/ar.1091140304

    Article  CAS  PubMed  Google Scholar 

  3. Dalton AJ, Felix MD (1954) Cytologic and cytochemical characteristics of the Golgi substance of epithelial cells of the epididymis–in situ, in homogenates and after isolation. Am J Anat 94:171–207. https://doi.org/10.1002/aja.1000940202

    Article  CAS  PubMed  Google Scholar 

  4. Geuze HJ (1999) A future for electron microscopy in cell biology? Trends Cell Biol 9:92–93. https://doi.org/10.1016/S0962-8924(98)01493-7

    Article  CAS  PubMed  Google Scholar 

  5. Heuser J (2002) Whatever happened to the ‘microtrabecular concept’? Biol Cell 94:561–596. https://doi.org/10.1016/S0248-4900(02)00013-8

    Article  PubMed  Google Scholar 

  6. Small JV (1981) Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J Cell Biol 91:695–705

    Article  CAS  Google Scholar 

  7. Maupin-Szamier P, Pollard TD (1978) Actin filament destruction by osmium tetroxide. J Cell Biol 77:837–852

    Article  CAS  Google Scholar 

  8. Dubochet J, McDowall AW, Menge B et al (1983) Electron microscopy of frozen-hydrated bacteria. J Bacteriol 155:381–390

    Article  CAS  Google Scholar 

  9. Dubochet J, McDowall AW (1981) Vitrification of pure water for Electron microscopy. J Microsc 124:3–4. https://doi.org/10.1111/j.1365-2818.1981.tb02483.x

    Article  Google Scholar 

  10. Dubochet J, Adrian M, Chang J-J et al (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228. https://doi.org/10.1017/S0033583500004297

    Article  CAS  PubMed  Google Scholar 

  11. Medalia O, Weber I, Frangakis AS et al (2002) Macromolecular architecture in eukaryotic cells visualized by Cryoelectron tomography. Science 298:1209–1213. https://doi.org/10.1126/science.1076184

    Article  CAS  PubMed  Google Scholar 

  12. Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature 308:32–36. https://doi.org/10.1038/308032a0

    Article  CAS  PubMed  Google Scholar 

  13. Grimm R, Singh H, Rachel R et al (1998) Electron tomography of ice-embedded prokaryotic cells. Biophys J 74:1031–1042. https://doi.org/10.1016/S0006-3495(98)74028-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Serwas D, Su TY, Roessler M et al (2017) Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans. J Cell Biol 216:1659–1671. https://doi.org/10.1083/jcb.201610070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Moor H (1987) In: Steinbrecht RA, Zierold K (eds) Theory and practice of high pressure freezing BT-cryotechniques in biological electron microscopy. Springer, Berlin, Heidelberg, pp 175–191

    Chapter  Google Scholar 

  17. Frank J (2006) Electron tomography-methods for three-dimensional visualization of structures in the cell. Springer-Verlag, New York

    Google Scholar 

  18. Lin J, Nicastro D (2018) Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science 360:eaar1968. https://doi.org/10.1126/science.aar1968

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Guichard P, Chretien D, Marco S, Tassin AM (2010) Procentriole assembly revealed by cryo-electron tomography. EMBO J 29:1565–1572. https://doi.org/10.1038/emboj.2010.45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Danev R, Buijsse B, Khoshouei M et al (2014) Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc Natl Acad Sci U S A 111:15635–15640. https://doi.org/10.1073/pnas.1418377111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. von Loeffelholz O, Papai G, Danev R et al (2018) Volta phase plate data collection facilitates image processing and cryo-EM structure determination. J Struct Biol 202:191–199. https://doi.org/10.1016/j.jsb.2018.01.003

    Article  CAS  Google Scholar 

  22. Glaeser RM (2008) Retrospective: radiation damage and its associated “information limitations”. J Struct Biol 163:271–276. https://doi.org/10.1016/j.jsb.2008.06.001

    Article  CAS  PubMed  Google Scholar 

  23. Talmon Y (1987) In: Steinbrecht RA, Zierold K (eds) Electron beam radiation damage to organic and biological cryospecimens BT-cryotechniques in biological electron microscopy. Springer, Berlin, Heidelberg, pp 64–84

    Google Scholar 

  24. Dierksen K, Typke D, Hegerl R et al (1992) Towards automatic electron tomography. Ultramicroscopy 40:71–87. https://doi.org/10.1016/0304-3991(92)90235-C

    Article  Google Scholar 

  25. Koster AJ, Chen H, Sedat JW, Agard DA (1992) Automated microscopy for electron tomography. Ultramicroscopy 46:207–227. https://doi.org/10.1016/0304-3991(92)90016-D

    Article  CAS  PubMed  Google Scholar 

  26. Baumeister W, Grimm R, Walz J (1999) Electron tomography of molecules and cells. Trends Cell Biol 9:81–85

    Article  CAS  Google Scholar 

  27. Xiong Q, Morphew MK, Schwartz CL et al (2009) CTF determination and correction for low dose tomographic tilt series. J Struct Biol 168:378–387. https://doi.org/10.1016/j.jsb.2009.08.016

    Article  PubMed  PubMed Central  Google Scholar 

  28. Fernández JJ, Li S, Crowther RA (2006) CTF determination and correction in electron cryotomography. Ultramicroscopy 106:587–596. https://doi.org/10.1016/j.ultramic.2006.02.004

    Article  CAS  PubMed  Google Scholar 

  29. Turoňová B, Schur FKM, Wan W, Briggs JAG (2017) Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4Å. J Struct Biol 199:187–195. https://doi.org/10.1016/j.jsb.2017.07.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Henderson R, Baldwin JM, Ceska TA et al (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:899–929. https://doi.org/10.1016/S0022-2836(05)80271-2

    Article  CAS  PubMed  Google Scholar 

  31. Fernandez J-J, Li S, Bharat TAM, Agard DA (2018) Cryo-tomography tilt-series alignment with consideration of the beam-induced sample motion. J Struct Biol 202:200–209. https://doi.org/10.1016/j.jsb.2018.02.001

    Article  PubMed  PubMed Central  Google Scholar 

  32. 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

    Article  PubMed  PubMed Central  Google Scholar 

  33. Grimm R, Koster AJ, Ziese U et al (1996) Zero-loss energy filtering under low-dose conditions using a post-column energy filter. J Microsc 183:60–68. https://doi.org/10.1046/j.1365-2818.1996.77441.x

    Article  CAS  Google Scholar 

  34. Langmore JP, Smith MF (1992) Quantitative energy-filtered electron microscopy of biological molecules in ice. Ultramicroscopy 46:349–373. https://doi.org/10.1016/0304-3991(92)90024-E

    Article  CAS  PubMed  Google Scholar 

  35. Schröder RR, Hofmann W, Ménétret J-F (1990) Zero-loss energy filtering as improved imaging mode in cryoelectronmicroscopy of frozen-hydrated specimens. J Struct Biol 105:28–34. https://doi.org/10.1016/1047-8477(90)90095-T

    Article  Google Scholar 

  36. McMullan G, Chen S, Henderson R, Faruqi AR (2009) Detective quantum efficiency of electron area detectors in electron microscopy. Ultramicroscopy 109:1126–1143. https://doi.org/10.1016/j.ultramic.2009.04.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Milazzo A-C, Cheng A, Moeller A et al (2011) Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy. J Struct Biol 176:404–408. https://doi.org/10.1016/j.jsb.2011.09.002

    Article  PubMed  PubMed Central  Google Scholar 

  38. Gilbert P (1972) Iterative methods for the three-dimensional reconstruction of an object from projections. J Theor Biol 36:105–117. https://doi.org/10.1016/0022-5193(72)90180-4

    Article  CAS  PubMed  Google Scholar 

  39. Radermacher M (2006) In: Frank J (ed) Weighted Back-projection methods BT-electron tomography: methods for three-dimensional visualization of structures in the cell. Springer, New York, NY, pp 245–273

    Google Scholar 

  40. Wan W, Briggs JAG (2016) Chapter thirteen-cryo-electron tomography and subtomogram averaging. In: Crowther RA (ed) Methods enzymol. Academic Press, New York, pp 329–367

    Google Scholar 

  41. Mahamid J, Pfeffer S, Schaffer M et al (2016) Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351:969–972. https://doi.org/10.1126/science.aad8857

    Article  CAS  PubMed  Google Scholar 

  42. Mühleip AW, Dewar CE, Schnaufer A et al (2017) In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits. Proc Natl Acad Sci U S A 114:992–997. https://doi.org/10.1073/pnas.1612386114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schur FKM, Hagen WJH, de Marco A, Briggs JAG (2013) Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging. J Struct Biol 184:394–400. https://doi.org/10.1016/j.jsb.2013.10.015

    Article  CAS  PubMed  Google Scholar 

  44. 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

    Article  PubMed  Google Scholar 

  45. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76. https://doi.org/10.1006/jsbi.1996.0013

    Article  CAS  PubMed  Google Scholar 

  46. Hampton CM, Strauss JD, Ke Z et al (2017) Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat Protoc 12:150–167. https://doi.org/10.1038/nprot.2016.168. http://www.nature.com/nprot/journal/v12/n1/abs/nprot.2016.168.html#supplementary-information

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the Office of Science of the US Department of Energy DE-AC02-O5CH11231 (K.M.D.), the Human Frontier Science Program fellowship LT000234/2018-L (D.S.) and UCB Start-up funds (K.M.D.).

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Daniel Serwas & Karen M. Davies

  2. Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Karen M. Davies

Authors
  1. Daniel Serwas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karen M. Davies
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Daniel Serwas or Karen M. Davies .

Editor information

Editors and Affiliations

  1. Howard Hughes Medical Institute Departments of Biological Chemistry and Physiology, University of California, Los Angeles, Los Angeles, CA, USA

    Tamir Gonen

  2. Chemical Engineering, School for Engineering of Matter, Transport and Energy; Center for Applied Structural Discovery Biodesign Institute, Arizona State University, Tempe, AZ, USA

    Brent L. Nannenga

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Serwas, D., Davies, K.M. (2021). Getting Started with In Situ Cryo-Electron Tomography. In: Gonen, T., Nannenga, B.L. (eds) cryoEM. Methods in Molecular Biology, vol 2215. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0966-8_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0966-8_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0965-1

  • Online ISBN: 978-1-0716-0966-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation