Skip to main content
SpringerLink
Log in

Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy

  • Protocol
  • First Online:
Protein Complex Assembly

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1764))

Abstract

Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.

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

Access this chapter

Log in via an institution
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 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

References

  1. Frank J (2009) Single-particle reconstruction of biological macromolecules in electron microscopy—30 years. Q Rev Biophys 42(3):139–158. https://doi.org/10.1017/S0033583509990059

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Kelly DF, Dukovski D, Walz T (2010a) A practical guide to the use of monolayer purification and affinity grids. Methods Enzymol 481:83–107. https://doi.org/10.1016/S0076-6879(10)81004-3

    Article  PubMed  CAS  Google Scholar 

  3. Kubalek EW, Le Grice S, Brown PO (1994) Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid. J Struct Biol 113(2):117–123. https://doi.org/10.1006/jsbi.1994.1039

    Article  PubMed  CAS  Google Scholar 

  4. Kelly DF, Dukovski D, Walz T (2008b) Monolayer purification: a rapid method for isolating protein complexes for single-particle electron microscopy. Proc Natl Acad Sci U S A 105(12):4703–4708. https://doi.org/10.1073/pnas.0800867105

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kelly DF, Abeyrathne PD, Dukovski D, Walz T (2008) The Affinity Grid: a pre-fabricated EM grid for monolayer purification. J Mol Biol 382(2):423–433. https://doi.org/10.1016/j.jmb.2008.07.023

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Kelly DF, Lake RJ, Middelkoop TC, Fan H-Y, Artavanis-Tsakonas S, Walz T (2010c) Molecular structure and dimeric organization of the notch extracellular domain as revealed by electron microscopy. PLoS One 5(5):e10532

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tanner JR, Degen K, Gilmore BL, Kelly DF (2012) Capturing RNA-dependent pathways for cryo-EM analysis. Comput Struct Biotechnol J 1(1):e201204003. https://doi.org/10.5936/csbj.201204003

    Article  PubMed  PubMed Central  Google Scholar 

  8. Kelly DF, Dukovski D, Walz T (2010b) Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy. J Mol Biol 400(4):675–681. https://doi.org/10.1016/j.jmb.2010.05.045

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Sharma G, Pallesen J, Das S, Grassucci R, Langlois R, Hampton CM et al (2013) Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome. J Struct Biol 181(2):190–194. https://doi.org/10.1016/j.jsb.2012.11.006

    Article  PubMed  CAS  Google Scholar 

  10. Kiss G, Chen X, Brindley MA, Campbell P, Afonso CL, Ke Z et al (2014) Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications. Microsc Microanal 20(1):164–174. https://doi.org/10.1017/S1431927613013937

    Article  PubMed  CAS  Google Scholar 

  11. Guimei Y, Kunpeng L, Pengwei H, Xi J, Wen J (2016) Antibody-based affinity cryoelectron microscopy at 2.6-Å resolution. Structure 24(11):1984–1990. https://doi.org/10.1016/j.str.2016.09.008

    Article  CAS  Google Scholar 

  12. Degen K, Dukes M, Tanner JR, Kelly DF (2012) The development of affinity capture devices—a nanoscale purification platform for biological in situ transmission electron microscopy. RSC Adv. https://doi.org/10.1039/c2ra01163h

  13. Gilmore BL, Showalter SP, Dukes MJ, Tanner JR, Demmert AC, McDonald SM, Kelly DF (2013a) Visualizing viral assemblies in a nanoscale biosphere. Lab Chip 13(2):216–219. https://doi.org/10.1039/c2lc41008g

    Article  PubMed  CAS  Google Scholar 

  14. Dukes MJ, Gilmore BL, Tanner JR, McDonald SM, Kelly DF (2013) In situ TEM of biological assemblies in liquid. J Vis Exp 82:50936. https://doi.org/10.3791/50936

    Article  CAS  Google Scholar 

  15. Tanner JR, Demmert AC, Dukes MJ (2013) Cryo-SiN—an alternative substrate to visualize active viral assemblies. J Analyt Mol Tech 1(1):6

    Google Scholar 

  16. Pohlmann ES, Patel K, Guo S, Dukes MJ, Sheng Z, Kelly DF (2015) Real-time visualization of nanoparticles interacting with glioblastoma stem cells. Nano Lett 15(4):2329–2335. https://doi.org/10.1021/nl504481k

    Article  PubMed  CAS  Google Scholar 

  17. Gilmore BL, Tanner JR, McKell AO, Boudreaux CE, Dukes MJ, McDonald SM, Kelly DF (2013b) Molecular surveillance of viral processes using silicon nitride membranes. Micromachines 4:90–102. https://doi.org/10.3390/mi4010090

    Article  Google Scholar 

  18. Winton CE, Gilmore BL, Demmert AC, Karageorge V, Sheng Z, Kelly DF (2016) A microchip platform for structural oncology applications. NPJ Breast Cancer 2. https://doi.org/10.1038/npjbcancer.2016.16

  19. Gilmore BL, Winton CE, Demmert AC, Tanner JR, Bowman S, Karageorge V et al (2015) A molecular toolkit to visualize native protein assemblies in the context of human disease. Sci Rep 5:14440. https://doi.org/10.1038/srep14440

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116(1):190–199

    Article  CAS  PubMed  Google Scholar 

  21. Scheres SHW (2012) A Bayesian view on cryo-EM structure determination. J Mol Biol 415(2):406–418. https://doi.org/10.1016/j.jmb.2011.11.010

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. He Y, Yan C, Fang J, Inouye C, Tjian R, Ivanov I, Nogales E (2016) Near-atomic resolution visualization of human transcription promoter opening. Nature 533:359–365. https://doi.org/10.1038/nature17970

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Brzovic PS, Rajagopal P, Hoyt DW, King MC, Klevit RE (2001) Structure of a BRCA1-BARD1 heterodimeric RING-RING complex. Nat Struct Biol 8(10):833–837. https://doi.org/10.1038/nsb1001-833

    Article  PubMed  CAS  Google Scholar 

  24. Williams RS, Green R, Glover JN (2001) Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1. Nat Struct Biol 8:838–842. https://doi.org/10.1038/nsb1001-838

    Article  PubMed  CAS  Google Scholar 

  25. Gilmore BL, Liang Y, Winton CE, Patel K, Karageorge V, Varano AC, Dearnaley W, Sheng Z, Kelly DF (2017) Molecular analysis of BRCA1 in human breast cancer cells under oxidative stress. Sci Rep 7:43435. https://doi.org/10.1038/srep43435

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by NIH/NCI grant R01CA193578 to D.F.K.

Author information

Authors and Affiliations

  1. Virginia Tech Carilion Research Institute, Roanoke, VA, USA

    Brian L. Gilmore, A. Cameron Varano, William Dearnaley, Yanping Liang, Bridget C. Marcinkowski & Deborah F. Kelly

  2. Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Blacksburg, VA, USA

    A. Cameron Varano

  3. Protochips, Inc., Applications Science, Raleigh, NC, USA

    Madeline J. Dukes

  4. Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA

    Deborah F. Kelly

Authors
  1. Brian L. Gilmore
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Cameron Varano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William Dearnaley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yanping Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bridget C. Marcinkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Madeline J. Dukes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Deborah F. Kelly
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Deborah F. Kelly .

Editor information

Editors and Affiliations

  1. MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom

    Joseph A. Marsh

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Gilmore, B.L. et al. (2018). Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy. In: Marsh, J. (eds) Protein Complex Assembly. Methods in Molecular Biology, vol 1764. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7759-8_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7759-8_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7758-1

  • Online ISBN: 978-1-4939-7759-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation