Skip to main content
SpringerLink
Log in

Studying Membrane Protein Structures by MicroED

  • Protocol
  • First Online:
Book cover Structure and Function of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2302))

Abstract

Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.

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 109.00
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 219.99
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. Gonen T, Cheng Y, Sliz P et al (2005) Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438:633–638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wisedchaisri G, Gonen T (2013) Phasing electron diffraction data by molecular replacement: strategy for structure determination and refinement. Methods Mol Biol 955:243–272

    Article  CAS  PubMed  Google Scholar 

  3. Oshima A, Tani K, Hiroaki Y et al (2007) Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule. Proc Natl Acad Sci 104:10034–10039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yonekura K, Kato K, Ogasawara M et al (2015) Electron crystallography of ultrathin 3D protein crystals: atomic model with charges. Proc Natl Acad Sci 112:3368–3373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Liu S, Gonen T (2018) MicroED structure of the NaK ion channel reveals a Na+ partition process into the selectivity filter. Commun Biol 1:38

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Nannenga BL, Shi D, Leslie AGW et al (2014) High-resolution structure determination by continuous-rotation data collection in MicroED. Nat Methods 11:927–930

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel https://doi.org/10.1073/pnas.2020010117

  8. MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP https://doi.org/10.1101/2020.09.27.316109

  9. Arnal F, Balladore JL, Soum G et al (1976) Calculation of the cross sections of electron interaction with matter. Ultramicroscopy 2:305–310

    Article  Google Scholar 

  10. Shi D, Nannenga BL, Iadanza MG et al (2013) Three-dimensional electron crystallography of protein microcrystals. elife 2013:e01345

    Article  CAS  Google Scholar 

  11. Nannenga BL, Shi D, Hattne J et al (2014) Structure of catalase determined by MicroED. elife 3:e03600

    Article  PubMed  PubMed Central  Google Scholar 

  12. Hattne J, Shi D, Glynn C et al (2018) Analysis of global and site-specific radiation damage in cryo-EM. Structure 26:759–766.e4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gallagher-Jones M, Glynn C, Boyer DR et al (2018) Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp. Nat Struct Mol Biol 25(2):131–134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Jones CG, Martynowycz MW, Hattne J et al (2018) The cryoEM method microED as a powerful tool for small molecule structure determination. ACS Cent Sci 4:1587–1592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Vergara S, Lukes DA, Martynowycz MW et al (2017) MicroED structure of Au 146 (p-MBA) 57 at subatomic resolution reveals a twinned FCC cluster. J Phys Chem Lett 8:5523–5530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. de l CMJ, Hattne J, Shi D et al (2017) Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED. Nat Methods 14:399–402

    Article  CAS  Google Scholar 

  17. Clabbers MTB, van GE, Wan W et al (2017) Protein structure determination by electron diffraction using a single three-dimensional nanocrystal. Acta Crystallogr D Struct Biol 73:738–748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Van GE, Clabbers MTB, Das PP et al (2016) Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector. Acta Crystallogr A Found Adv 72:236–242

    Article  CAS  Google Scholar 

  19. Shi D, Nannenga BL, de l CMJ et al (2016) The collection of MicroED data for macromolecular crystallography. Nat Protoc 11:895–904

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hattne J, Reyes FE, Nannenga BL et al (2015) MicroED data collection and processing. Acta Crystallogr A Found Adv 71:353–360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Caffrey M (2003) Membrane protein crystallization. J Struct Biol 142:108–132

    Article  CAS  PubMed  Google Scholar 

  22. Petsko GA (1975) Protein crystallography at sub-zero temperatures: cryo-protective mother liquors for protein crystals. J Mol Biol 96:381–392

    Article  CAS  PubMed  Google Scholar 

  23. Kriminski S, Kazmierczak M, Thorne RE (2003) Heat transfer from protein crystals: implications for flash-cooling and X-ray beam heating. Acta Crystallogr D Biol Crystallogr 59:697–708

    Article  CAS  PubMed  Google Scholar 

  24. Marko M, Hsieh C, Schalek R et al (2007) Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat Methods 4:215–217

    Article  CAS  PubMed  Google Scholar 

  25. Rigort A, Bauerlein FJB, Villa E et al (2012) Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci 109:4449–4454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schertel A, Snaidero N, Han H-M et al (2013) Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens. J Struct Biol 184:355–360

    Article  CAS  PubMed  Google Scholar 

  27. Giannuzzi LA, Stevie FA (1999) A review of focused ion beam milling techniques for TEM specimen preparation. Micron 30:197–204

    Article  Google Scholar 

  28. Sergey G, Denis K, Ava H et al (2018) Oxygen plasma focused ion beam scanning electron microscopy for biological samples. bioRxiv 2018:457820. https://doi.org/10.1101/457820

    Article  CAS  Google Scholar 

  29. Martynowycz MW, Zhao W, Hattne J et al (2019) Collection of continuous rotation microED data from ion beam-milled crystals of any size. Structure 27:545–548.e2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Martynowycz MW, Zhao W, Hattne J et al (2019) Qualitative analyses of polishing and precoating FIB milled crystals for microED. Structure 27:1594–1600.e2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Duyvesteyn HME, Kotecha A, Ginn HM et al (2018) Machining protein microcrystals for structure determination by electron diffraction. Proc Natl Acad Sci 115:9569–9573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li X, Zhang S, Zhang J et al (2018) In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction. Biophys Rep 4:339–347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhou H, Luo Z, Li X (2019) Using focus ion beam to prepare crystal lamella for electron diffraction. J Struct Biol 205(3):59–64

    Article  CAS  PubMed  Google Scholar 

  34. Martynowycz M, Glynn C, Miao J, et al (2017) MicroED structures from micrometer thick protein crystals. https://doi.org/10.1101/152504

  35. Latychevskaia T, Abrahams JP (2019) Inelastic scattering and solvent scattering reduce dynamical diffraction in biological crystals. Acta Crystallogr B Struct Sci Cryst Eng Mater 75:523–531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Clabbers MTB, Abrahams JP (2018) Electron diffraction and three-dimensional crystallography for structural biology. Crystallogr Rev 24:176–204

    Article  CAS  Google Scholar 

  37. Clabbers MTB, Gruene T, van Genderen E et al (2019) Reducing dynamical electron scattering reveals hydrogen atoms. Acta Crystallogr A Found Adv 75:82–93

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Flot D, Mairs T, Giraud T et al (2010) The ID23-2 structural biology microfocus beamline at the ESRF. J Synchrotron Radiat 17:107–118

    Article  CAS  PubMed  Google Scholar 

  39. Palatinus L, Brázda P, Boullay P et al (2017) Hydrogen positions in single nanocrystals revealed by electron diffraction. Science 355:166–169

    Article  CAS  PubMed  Google Scholar 

  40. Brázda P, Palatinus L, Babor M (2019) Electron diffraction determines molecular absolute configuration in a pharmaceutical nanocrystal. Science 364:667–669

    Article  PubMed  CAS  Google Scholar 

  41. Gruene T, Wennmacher JTC, Zaubitzer C et al (2018) Rapid structure determination of microcrystalline molecular compounds using electron diffraction. Angew Chem Int Ed 57:16313–16317

    Article  CAS  Google Scholar 

  42. Spurlino JC, Lu GY, Quiocho FA (1991) The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. J Biol Chem 266:5202–5219

    Article  CAS  PubMed  Google Scholar 

  43. Yonekura K, Maki-Yonekura S (2016) Refinement of cryo-EM structures using scattering factors of charged atoms. J Appl Crystallogr 49:1517–1523

    Article  CAS  Google Scholar 

  44. Alam A, Jiang Y (2009) Structural analysis of ion selectivity in the NaK channel. Nat Struct Mol Biol 16:35–41

    Article  CAS  PubMed  Google Scholar 

  45. Alam A, Jiang Y (2009) High-resolution structure of the open NaK channel. Nat Struct Mol Biol 16:30–34

    Article  CAS  PubMed  Google Scholar 

  46. Rodriguez JA, Gonen T (2016) High-resolution macromolecular structure determination by microED, a cryo-EM method. Elsevier, Inc.

    Book  Google Scholar 

  47. Nannenga BL, Gonen T (2014) Protein structure determination by microED. Curr Opin Struct Biol 27:24–31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kabsch W (2010) Xds. Acta Crystallogr D Biol Crystallogr 66:125–132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Leslie AGW, Powell HR (2007) Processing diffraction data with mosflm. https://doi.org/10.1007/978-1-4020-6316-9_4

  50. Battye TGG, Kontogiannis L, Johnson O et al (2011) iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67:271–281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Winter G, Waterman DG, Parkhurst JM et al (2018) DIALS: implementation and evaluation of a new integration package. Acta Crystallogr D Struct Biol 74:85–97

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Clabbers MTB, Gruene T, Parkhurst JM et al (2018) Electron diffraction data processing with DIALS. Acta Crystallogr D Struct Biol 74:506–518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Sheldrick GM (2015) SHELXT – integrated space-group and crystal-structure determination. Acta Crystallogr A Found Crystallogr 71:3–8

    Article  CAS  Google Scholar 

  54. Schneider TR, Sheldrick GM (2002) Substructure solution with SHELXD. Acta Crystallogr D Biol Crystallogr 58:1772–1779

    Article  PubMed  CAS  Google Scholar 

  55. McCoy AJ, Grosse-Kunstleve RW, Adams PD et al (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Crystallogr 30:1022–1025

    Article  CAS  Google Scholar 

  57. Sawaya MR, Rodriguez J, Cascio D et al (2016) Ab initio structure determination from prion nanocrystals at atomic resolution by microED. Proc Natl Acad Sci 113:11232–11236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Martynowycz MW, Hattne J, Gonen T (2020) Experimental phasing of microED data using radiation damage. Structure 28:458–464.e2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The Gonen lab is supported by funds from the Howard Hughes Medical Institute and the National Institutes of Health P41GM136508. We would like to thank the members of the Jensen and Gonen laboratories for helpful discussions.

Author information

Authors and Affiliations

  1. Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA

    Michael W. Martynowycz & Tamir Gonen

  2. Department of Physiology, University of California Los Angeles, Los Angeles, CA, USA

    Michael W. Martynowycz & Tamir Gonen

  3. Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, CA, USA

    Michael W. Martynowycz & Tamir Gonen

Authors
  1. Michael W. Martynowycz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tamir Gonen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tamir Gonen .

Editor information

Editors and Affiliations

  1. School of Biological Sciences and School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Ingeborg Schmidt-Krey

  2. School of Physics, Georgia Institute of Technology, Atlanta, GA, USA

    James C. Gumbart

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

Martynowycz, M.W., Gonen, T. (2021). Studying Membrane Protein Structures by MicroED. In: Schmidt-Krey, I., Gumbart, J.C. (eds) Structure and Function of Membrane Proteins. Methods in Molecular Biology, vol 2302. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1394-8_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1394-8_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1393-1

  • Online ISBN: 978-1-0716-1394-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation