Skip to main content
SpringerLink
Log in

In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography

  • Protocol
  • First Online:
Bacterial Protein Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

Abstract

The unique property of electron cryotomography (ECT) is its capability to resolve the structure of macromolecular machines in their cellular context. The integration of ECT data with high-resolution structures of purified subcomplexes and live-cell fluorescence light microscopy can generate pseudo-atomic models that lead to a mechanistic understanding across size and time scales. Recent advances in electron detection, sample thinning, data acquisition, and data processing have significantly enhanced the applicability and performance of ECT. Here we describe a detailed workflow for an ECT experiment, including cell culture, vitrification, data acquisition, data reconstruction, tomogram analysis, and subtomogram averaging. This protocol provides an entry point to the technique for students and researchers and indicates the many possible variations arising from specific target properties and the available instrumentation.

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

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Costa TD, Felisberto-Rodrigues C, Meir A, Prevost MS, Redzej A, Trocker M, Waksman G (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13:343–359

    Google Scholar 

  2. Gan L, Jensen GJ (2012) Electron tomography of cells. Q Rev Biophys 45:27–56

    Article  CAS  PubMed  Google Scholar 

  3. Harapin J, Eibauer M, Medalia O (2013) Structural analysis of supramolecular assemblies by cryo-electron tomography. Structure 21:1522–1530

    Article  CAS  PubMed  Google Scholar 

  4. Briggs JAG (2013) Structural biology in situ—the potential of subtomogram averaging. Curr Opin Struct Biol 23:261–267

    Article  CAS  PubMed  Google Scholar 

  5. Lučić V, Rigort A, Baumeister W (2013) Cryo-electron tomography: the challenge of doing structural biology in situ. J Cell Biol 202:407–419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 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

    Article  CAS  PubMed  Google Scholar 

  7. Pilhofer M, Ladinsky MS, McDowall AW, Jensen GJ (2010) Bacterial TEM. Methods Cell Biol 96:21–45

    Article  PubMed  Google Scholar 

  8. Schur FKM, Obr M, Hagen WJH, Wan W, Jakobi AJ, Kirkpatrick JM, Sachse C, Kräuslich HG, Briggs JAG (2016) An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science 353:506–508

    Article  CAS  PubMed  Google Scholar 

  9. Basler M, Pilhofer M, Henderson GP, Jensen GJ, Mekelanos JJ (2012) Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483:182–186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Abrusci P, Vergara-Irigaray M, Johnson S, Beeby MD, Hendrixson DR, Roversi P, Friede ME, Deane JE, Jensen GJ, Tang CM, Lea SM (2013) Architecture of the major component of the type III secretion system export apparatus. Nat Struct Mol Biol 20:99–104

    Article  CAS  PubMed  Google Scholar 

  11. Kawamoto A, Morimoto YV, Miyata T, Minamino T, Hughes KT, Kato T, Namba K (2013) Common and distinct structural features of Salmonella injectisome and flagellar basal body. Sci Rep 3:3396

    Article  Google Scholar 

  12. Kudryashev M, Stenta M, Schmelz S, Amstutz M, Wiesand U, Castaño-Diez D, Degiacomi MT, Münnich S, Bleck CKE, Kowal J, Diepold A, Heinz DW, Dal Peraro M, Cornelis GR, Stahlberg H (2013) In situ structural analysis of the Yersinia enterocolitica injectisome. elife 2:e00792

    Article  PubMed  PubMed Central  Google Scholar 

  13. Nans A, Saibil HR, Hayward RD (2014) Pathogen-host reorganization during Chlamydia invasion revealed by cryo-electron tomography. Cell Microbiol 16:1457–1472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pilhofer M, Aistleitner K, Ladinsky MS, König L, Horn M, Jensen GJ (2014) Architecture and host interface of environmental chlamydiae revealed by electron cryotomography. Environ Microbiol 16:417–429

    Article  CAS  PubMed  Google Scholar 

  15. Radics J, Königsmaier L, Marlovits TC (2014) Structure of a pathogenic type 3 secretion system in action. Nat Struct Mol Biol 21:82–87

    Article  CAS  PubMed  Google Scholar 

  16. Shikuma NJ, Pilhofer M, Weiss GL, Hadfield MG, Jensen GJ, Newman DK (2014) Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343:529–533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hu B, Morado DR, Margolin W, Rohda JR, Arizmendi O, Picking WL, Picking WD, Liu J (2015) Visualization of the type III secretion sorting platform of Shigella flexneri. Proc Natl Acad Sci U S A 112:1047–1052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kudryashev M, Diepold A, Amstutz M, Armitage JP, Stahlberg H, Cornelis GR (2015) Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation. Mol Microbiol 95:875–884

    Article  CAS  PubMed  Google Scholar 

  19. Nans A, Kudryashev M, Saibil HR, Hayward RD (2015) Structure of a bacterial type III secretion system in contact with a host membrane in situ. Nat Commun 6:10114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chang YW, Rettberg LA, Treuner-Lange A, Iwasa J, Segaard-Anderson L, Jensen GJ (2016) Architecture of the type IVa pilus machine. Science 351:1165–1172

    Article  CAS  Google Scholar 

  21. Kühlbrandt W (2014) The resolution revolution. Science 343:1443–1444

    Article  PubMed  Google Scholar 

  22. McMullan G, Faruqi AR, Clare D, Henderson R (2014) Comparison of optimal performance at 300 keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147:156–163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Campbell MG, Cheng A, Brilot AF, Moeller A, Lyumkis D, Veesler D, Pan J, Harrison SC, Potter CS, Carragher B, Grigorieff N (2012) Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20:1823–1828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Danev R, Buijsse B, Khoshouei M, Plitzko JM, Baumeister W (2014) Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc Natl Acad Sci U S A 111:15635–15640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Briegel A, Chen S, Koster AJ, Plitzko JM, Schwartz CL, Jensen GJ (2010) Correlated light and electron cryo-microscopy. Methods Enzymol 481:317–341

    Article  PubMed  Google Scholar 

  26. Chang YW, Chen S, Tocheva EI, Treuner-Lange A, Löbach S, Søgaard-Anderson L, Jensen GJ (2014) Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat Methods 11:737–739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rigort A, Bäuerlein FJB, Villa E, Eibauer M, Laugks T, Baumeister W, Plitzko JM (2012a) Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci U S A 109:4449–4454

    Article  PubMed  PubMed Central  Google Scholar 

  28. Hagen WJH, Wan W, Briggs JAG (2016) Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J Struct Biol

    Google Scholar 

  29. Bharat TAM, Russo CJ, Löwe J, Passmore LA, Scheres SHW (2015) Advances in single-particle electron cryomicroscopy structure determination applied to sub-tomogram averaging. Structure 23:1743–1753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Suloway C, Pulokas J, Fellmann D, Cheng A, Guerra F, Quispe J, Stagg S, Potter CS, Carragher B (2005) Automated molecular microscopy: the new Leginon system. J Struct Biol 151:41–60

    Article  CAS  PubMed  Google Scholar 

  31. Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

    Article  PubMed  Google Scholar 

  32. Zheng SQ, Keszthelyi B, Branlund E, Lyle JM, Braunfeld MB, Sedat JW, Agard DA (2007) UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction. J Struct Biol 157:138–147

    Article  CAS  PubMed  Google Scholar 

  33. Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB, Gubbens S, Agard DA, Cheng Y (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mastronarde DN (2008) Correction for non-perpendicularity of beam and tilt axis in tomographic reconstructions with the IMOD package. J Microsc 230:212–217

    Article  CAS  PubMed  Google Scholar 

  35. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76

    Article  CAS  PubMed  Google Scholar 

  36. Kunz M, Frangakis AS (2014) Super-sampling SART with ordered subsets. J Struct Biol 188:107–115

    Article  PubMed  Google Scholar 

  37. Chen Y, Förster F (2014) Iterative reconstruction of cryo-electron tomograms using nonuniform fast Fourier transforms. J Struct Biol 185:309–316

    Article  PubMed  Google Scholar 

  38. Agulleiro JI, Fernandez JJ (2015) Tomo3D 2.0—exploitation of advanced vector eXtensions (AVX) for 3D reconstruction. J Struct Biol 189:147–152

    Article  PubMed  Google Scholar 

  39. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  PubMed  Google Scholar 

  40. Castaño-Díez D, Kudryashev M, Arheit M, Stahlberg H (2012) Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J Struct Biol 178:139–151

    Article  PubMed  Google Scholar 

  41. Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME, McIntosh JR (2006) The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313:944–948

    Article  CAS  PubMed  Google Scholar 

  42. Tivol WF, Briegel A, Jensen GJ (2008) An improved cryogen for plunge freezing. Microsc Microanal 14:375–379

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Iancu CV, Tivol WF, Schooler JB, Dias PD, Henderson GP, Murphy GE, Wright ER, Li Z, Yu Z, Briegel A, Gan L, He Y, Jensen GJ (2006) Electron cryotomography sample preparation using the Vitrobot. Nat Protoc 1:2813–2819

    Article  CAS  PubMed  Google Scholar 

  44. Chen S, McDowall A, Dobro MJ, Briegel A, Ladinsky M, Shi J, Tocheva EI, Beeby M, Pilhofer M, Ding HJ, Li Z, Gan L, Morris DM, Jensen GJ (2010) Electron cryotomography of bacterial cells. J Vis Exp 39:e1943

    Google Scholar 

  45. Brüggeller P, Mayer E (1980) Complete vitrification in pure liquid water and dilute aqueous solutions. Nature 288:569–571

    Article  Google Scholar 

  46. Radermacher M (2007) Weighted back-projection methods. In: Electron tomography. Springer, New York

    Google Scholar 

  47. Wolf D, Lubk A, Lichte H (2014) Weighted simultaneous iterative reconstruction technique for single-axis tomography. Ultramicroscopy 136:15–25

    Article  CAS  PubMed  Google Scholar 

  48. Briegel A, Pilhofer M, Mastronarde DN, Jensen GJ (2013) The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards. J Struct Biol 183:95–98

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Rigort A, Günther D, Hegerl R, Baum D, Weber B, Prohaska S, Medalia O, Baumeister W, Hege HC (2012b) Automated segmentation of electron tomograms for a quantitative description of actin filament networks. J Struct Biol 177:135–144

    Article  CAS  PubMed  Google Scholar 

  50. Volkmann N (2002) A novel three-dimensional variant of the watershed transform for segmentation of electron density maps. J Struct Biol 138:123–129

    Article  CAS  PubMed  Google Scholar 

  51. Baker ML, Yu Z, Chiu W, Bajaj C (2006) Automated segmentation of molecular subunits in electron cryomicroscopy density maps. J Struct Biol 156:432–441

    Article  CAS  PubMed  Google Scholar 

  52. Heumann JM, Hoenger A, Mastronarde DN (2011) Clustering and variance maps for cryo-electron tomography using wedge-masked differences. J Struct Biol 175:288–299

    Article  PubMed  PubMed Central  Google Scholar 

  53. Galán JE, Curtiss R (1990) Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect Immun 58:1879–1885

    PubMed  PubMed Central  Google Scholar 

  54. Basler M, Ho BT, Mekalanos JJ (2013) Tit-for-Tat: Type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152:884–894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Eichelberg K, Galán JE (1999) Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and hilA. Infect Immun 67:4099–4105

    PubMed  PubMed Central  CAS  Google Scholar 

  56. Farley MM, Hu B, Margolin W, Liu J (2016) Minicells, back in fashion. J Bacteriol 198:1186–1195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Briegel A, Wong ML, Hodges HL, Oikonomou CM, Piasta KN, Harris MJ, Fowler DJ, Thompson LK, Falke JJ, Kiessling LL, Jensen GJ (2014) New insights into bacterial chemoreceptor Array structure and assembly from electron cryotomography. Biochemistry 53:1575–1585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Crowther RA, DeRosier DJ, Klug A (1970) The reconstruction of a three-dimensional structure from projections and its application to electron microscopy. Proc R Soc A 317:319–340

    Article  Google Scholar 

  59. Wan W, Briggs JAG (2016) Cryo-electron tomography and subtomogram averaging. Methods Enzymol 579:329–367

    Article  CAS  PubMed  Google Scholar 

  60. Pfeffer S, Burbaum L, Unverdorben P, Pech M, Chen Y, Zimmermann R, Beckman R, Förster F (2015) Structure of the native Sec61 protein-conducting channel. Nat Commun 6:8403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Amat F, Moussavi F, Comolli LR, Elidan G, Downing KH, Horowitz M (2008) Markov random field based automatic image alignment for electron tomography. J Struct Biol 161:260–275

    Article  PubMed  Google Scholar 

  62. Morado DR, Hu B, Liu J (2016) Using Tomoauto: a protocol for high-throughput automated cryo-electron tomography. J Vis Exp 107:e53608

    Google Scholar 

Download references

Acknowledgments

We thank D. Böck, R. Kooger, and P. Szwedziak for comments on the manuscript. G. L. Weiss was supported by a Boehringer Ingelheim Fonds PhD Fellowship. The Pilhofer Lab is supported by grants from ETH Zürich, the European Research Council, the Swiss National Science Foundation, and the Helmut Horten Foundation.

Author information

Authors and Affiliations

  1. Department of Biology, ETH Zürich, Institute of Molecular Biology and Biophysics, Otto-Stern-Weg 5, 8093, Zürich, Switzerland

    Gregor L. Weiss, João M. Medeiros & Martin Pilhofer

Authors
  1. Gregor L. Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. João M. Medeiros
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Pilhofer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martin Pilhofer .

Editor information

Editors and Affiliations

  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Weiss, G.L., Medeiros, J.M., Pilhofer, M. (2017). In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_27

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7033-9_27

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7031-5

  • Online ISBN: 978-1-4939-7033-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation