Nanoscale Visualization of Bacterial Microcompartments Using Atomic Force Microscopy

  • Jorge Rodriguez-Ramos
  • Matthew Faulkner
  • Lu-Ning Liu
Part of the Methods in Molecular Biology book series (MIMB, volume 1814)


Bacterial microcompartments (BMCs) are polyhedral protein organelles in many prokaryotes, playing significant roles in metabolic enhancement. Due to their self-assembly and modularity nature, BMCs have gained increased interest in recent years, with the intent of constructing new nanobioreactors and scaffolding to promote cellular metabolisms and molecule delivery. In this chapter, we describe the technique of atomic force microscopy (AFM) as a method to study the self-assembly dynamics and physical properties of BMCs. We focus on the sample preparation, the measurement procedure, and the data analysis for high-speed AFM imaging and nanoindentation-based spectroscopy, which were used to determine the assembly dynamics of BMC shell proteins and the nanomechanics of intact BMC structures, respectively. The described methods could be applied to the study of other types of self-assembling biological organelles.

Key words

Atomic force microscopy High-speed AFM Force spectroscopy Nanoindentation Bacterial microcompartment Carboxysome Self-assembly Nanomechanics 



This work was supported by a Royal Society University Research Fellowship (UF120411), a Royal Society Research grant for University Research Fellowship (RG130442), a Royal Society Challenge grant (CH160004), a Biotechnology and Biological Sciences Research Council grant (BB/R003890/1), and a Biotechnology and Biological Sciences Research Council grant (BB/M024202/1). We acknowledge the Liverpool Centre for Cell Imaging for technical assistance and access to confocal/TIRF microscopes (Biotechnology and Biological Sciences Research Council, BB/M012441/1).


  1. 1.
    Sutter M, Faulkner M, Aussignargues C, Paasch BC, Barrett S, Kerfeld CA, Liu L-N (2016) Visualization of bacterial microcompartment facet assembly using high-speed atomic force microscopy. Nano Lett 16(3):1590–1595. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Klein MG, Zwart P, Bagby SC, Cai F, Chisholm SW, Heinhorst S, Cannon GC, Kerfeld CA (2009) Identification and structural analysis of a novel carboxysome shell protein with implications for metabolite transport. J Mol Biol 392(2):319–333. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Tanaka S, Kerfeld CA, Sawaya MR, Cai F, Heinhorst S, Cannon GC, Yeates TO (2008) Atomic-level models of the bacterial carboxysome shell. Science 319(5866):1083–1086. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kerfeld CA, Sawaya MR, Tanaka S, Nguyen CV, Phillips M, Beeby M, Yeates TO (2005) Protein structures forming the shell of primitive bacterial organelles. Science 309(5736):936–938. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Yeates TO, Crowley CS, Tanaka S (2010) Bacterial microcompartment organelles: protein shell structure and evolution. Annu Rev Biophys 39:185–205. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Rae BD, Long BM, Badger MR, Price GD (2013) Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol Mol Biol Rev 77(3):357–379CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Behrenfeld MJ, Randerson JT, McClain CR, Feldman GC, Los SO, Tucker CJ, Falkowski PG, Field CB, Frouin R, Esaias WE, Kolber DD, Pollack NH (2001) Biospheric primary production during an ENSO transition. Science 291(5513):2594–2597. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ando T (2017) Directly watching biomolecules in action by high-speed atomic force microscopy. Biophys Rev 9:421. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Uchihashi T, Scheuring S (2017) Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes. Biochim Biophys Acta 1862:229. CrossRefPubMedGoogle Scholar
  10. 10.
    Preiner J, Horner A, Karner A, Ollinger N, Siligan C, Pohl P, Hinterdorfer P (2015) High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties. Nano Lett 15(1):759–763. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Uchihashi T, Kodera N, Ando T (2012) Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat Protoc 7(6):1193–1206. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sicard D, Fredenburgh LE, Tschumperlin DJ (2017) Measured pulmonary arterial tissue stiffness is highly sensitive to AFM indenter dimensions. J Mech Behav Biomed Mater 74:118–127. CrossRefPubMedGoogle Scholar
  13. 13.
    Ramos JR, Pabijan J, Garcia R, Lekka M (2014) The softening of human bladder cancer cells happens at an early stage of the malignancy process. Beilstein J Nanotechnol 5(1):447–457. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Marchetti M, Wuite G, Roos WH (2016) Atomic force microscopy observation and characterization of single virions and virus-like particles by nano-indentation. Curr Opin Virol 18:82–88CrossRefPubMedGoogle Scholar
  15. 15.
    Mateu MG (2012) Mechanical properties of viruses analyzed by atomic force microscopy: a virological perspective. Virus Res 168(1–2):1–22. CrossRefPubMedGoogle Scholar
  16. 16.
    Liu LN, Scheuring S (2013) Investigation of photosynthetic membrane structure using atomic force microscopy. Trends Plant Sci 18(5):277–286. CrossRefPubMedGoogle Scholar
  17. 17.
    Liu LN, Duquesne K, Oesterhelt F, Sturgis JN, Scheuring S (2011) Forces guiding assembly of light-harvesting complex 2 in native membranes. Proc Natl Acad Sci U S A 108(23):9455–9459CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sutter M, Greber B, Aussignargues C, Kerfeld CA (2017) Assembly principles and structure of a 6.5-MDa bacterial microcompartment shell. Science 356(6344):1293–1297. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lassila JK, Bernstein SL, Kinney JN, Axen SD, Kerfeld CA (2014) Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function. J Mol Biol 426(11):2217–2228. CrossRefPubMedGoogle Scholar
  20. 20.
    Roos WH (2011) How to perform a nanoindentation experiment on a virus. In: Peterman EJG, Wuite GJL (eds) Single molecule analysis: methods and protocols. Humana Press, Totowa, NJ, pp 251–264. CrossRefGoogle Scholar
  21. 21.
    Faulkner M, Rodriguez-Ramos J, Dykes GF, Owen SV, Casella S, Simpson DM, Beynon RJ, Liu L-N (2017) Direct characterization of the native structure and mechanics of cyanobacterial carboxysomes. Nanoscale 9(30):10662–10673. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Liu LN, Sturgis JN, Scheuring S (2011) Native architecture of the photosynthetic membrane from Rhodobacter veldkampii. J Struct Biol 173(1):138–145. CrossRefPubMedGoogle Scholar
  23. 23.
    Liu LN, Duquesne K, Sturgis JN, Scheuring S (2009) Quinone pathways in entire photosynthetic chromatophores of Rhodospirillum photometricum. J Mol Biol 393(1):27–35. CrossRefPubMedGoogle Scholar
  24. 24.
    Miller EJ, Trewby W, Farokh Payam A, Piantanida L, Cafolla C, Voitchovsky K (2016) Sub-nanometer resolution imaging with amplitude-modulation atomic force microscopy in liquid. J Vis Exp 118:54924Google Scholar
  25. 25.
    Kumar S, Cartron ML, Mullin N, Qian P, Leggett GJ, Hunter CN, Hobbs JK (2017) Direct Imaging of Protein Organization in an intact bacterial organelle using high-resolution atomic force microscopy. ACS Nano 11(1):126–133. CrossRefPubMedGoogle Scholar
  26. 26.
    Scheuring S, Nevo R, Liu LN, Mangenot S, Charuvi D, Boudier T, Prima V, Hubert P, Sturgis JN, Reich Z (2014) The architecture of Rhodobacter sphaeroides chromatophores. Biochim Biophys Acta 1837(8):1263–1270. CrossRefPubMedGoogle Scholar
  27. 27.
    Sader JE, Chon JWM, Mulvaney P (1999) Calibration of rectangular atomic force microscope cantilevers. Rev Sci Instrum 70(10):3967–3969. CrossRefGoogle Scholar
  28. 28.
    Cook S, Schaffer TE, Chynoweth KM, Wigton M, Simmonds RW, Lang KM (2006) Practical implementation of dynamic methods for measuring atomic force microscope cantilever spring constants. Nanotechnology 17(9):2135–2145. CrossRefGoogle Scholar
  29. 29.
    Ramos JR (2014) Tip radius preservation for high resolution imaging in amplitude modulation atomic force microscopy. Appl Phys Lett 105(4):043111. CrossRefGoogle Scholar
  30. 30.
    Carrasco C, Carreira A, Schaap IAT, Serena PA, Gómez-Herrero J, Mateu MG, de Pablo PJ (2006) DNA-mediated anisotropic mechanical reinforcement of a virus. Proc Natl Acad Sci 103(37):13706–13711. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Jorge Rodriguez-Ramos
    • 1
  • Matthew Faulkner
    • 1
  • Lu-Ning Liu
    • 1
  1. 1.Institute of Integrative BiologyUniversity of LiverpoolLiverpoolUK

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