Advertisement

Automation of Data Acquisition in Electron Crystallography

  • Anchi Cheng
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

Abstract

General considerations for using automation software for acquiring high-resolution images of 2D crystals under low-dose conditions are presented. Protocol modifications specific to this application in Leginon are provided.

Key words

Automation Electron crystallography Electron microscopy Leginon 

Notes

Acknowledgments

This work is supported by National Institutes of Health (NIH) through the National Center for Research Resources (NCRR) P41 program (Grants RR17573 and RR023093). I would like to thank Bridget Carragher for valuable comments and editing of the manuscript.

References

  1. 1.
    Carragher B, Kisseberth N, Kriegman D, Milligan RA, Potter CS, Pulokas J, Reilein A (2000) Leginon: an automated system for acquisition of images from vitreous ice specimens. J Struct Biol 132:33–45PubMedCrossRefGoogle Scholar
  2. 2.
    Lei J, Frank J (2005) Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J Struct Biol 150:69–80PubMedCrossRefGoogle Scholar
  3. 3.
    Marsh MP, Chang JT, Booth CR, Liang NL, Schmid MF, Chiu W (2007) Modular software platform for low-dose electron microsopy and tomography. J Microsc 228:384–389PubMedCrossRefGoogle Scholar
  4. 4.
    Nickell S, Beck F, Korinek A, Mihalache O, Baumeister W, Plitzko JM (2007) Automated cryoelectron microscopy of “single particles” applied to the 26S proteasome. FEBS Lett 581:2751–2756PubMedCrossRefGoogle Scholar
  5. 5.
    Shi J, Williams DR, Stewart PL (2008) A Script-assisted microscopy (SAM) package to improve data acquisition rates on FEI Tecnai electron microscopes equipped with Gatan CCD cameras. J Struct Biol 164:166–169PubMedCrossRefGoogle Scholar
  6. 6.
    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 151:41–60PubMedCrossRefGoogle Scholar
  7. 7.
    Yoshioka C, Pulokas J, Fellmann D, Potter CS, Milligan RA, Carragher B (2007) Automation of random conical tilt and orthogonal tilt data collection using feature-based correlation. J Struct Biol 159:335–346PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang J, Nakamura N, Shimizu Y, Liang N, Liu X, Jakana J, Marsh MP, Booth CR, Shinkawa T, Nakta M, Chiu W (2009) JADAS: a customizable automated data acquisition system and its application to ice-embedded single particles. J Struct Biol 165:1–9PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang P, Beatty A, Milne JLS, Subramaniam S (2001) Automated data collection with a Tecnai 12 electron microscope: applications for molecular imaging by cryomicroscopy. J Struct Biol 135:251–261PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang P, Borgnia MJ, Mooney P, Shi D, Pan M, O’Herron P, Mao A, Brogan D, Milne JLS, Subramaniam S (2003) Automated image acquisition and processing using a new generation of 4K x 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies. J Struct Biol 143:135–144PubMedCrossRefGoogle Scholar
  11. 11.
    FEI (2005) Advanced Tecnai TEM software for easy acquisition, reconstruction and visualization, Technical Note, FEI softwareGoogle Scholar
  12. 12.
    Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51PubMedCrossRefGoogle Scholar
  13. 13.
    Nickell S, Förster F, Linaroudis A, Net WD, Beck F, Hegeri R, Baumeister W, Plitzko JM (2005) TOM software toolbox: acquisition and analysis for electron tomography. J Struct Biol 149:227–234PubMedCrossRefGoogle Scholar
  14. 14.
    Cheng A, Leung A, Fellmann D, Quispe J, Suloway C, Pulokas J, Abeyrathne PD, Lam JS, Carragher B, Potter CS (2007) Towards automated screening of two-dimensional crystals. J Struct Biol 160:263–404CrossRefGoogle Scholar
  15. 15.
    Hu M, Vink M, Kim C, Derr K, Koss J, D’Amico K, Cheng A, Pulokas J, Ubarretxena-Belandia I, Stokes D (2010) Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins. J Struct Biol 171:102–110PubMedCrossRefGoogle Scholar
  16. 16.
    Nakamura N, Fujiyoshi Y, Mitsuoka K, Murata K, Shinkawa T (2003) An image evaluation system coupled with Auto-TEM. Microsc Microanal 9(Suppl 2):1038CD–1039CDGoogle Scholar
  17. 17.
    Coudray N, Hermann G, Caujolle-Bert D, Karathanou A, Erne-Brand F, Bruessler J-L, Daum P, Plitzko JM, Chami M, Mueller U, Kihl H, Urban J-P, Engel A, Remigy W (2011) Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis. J Struct Biol 173:365–374PubMedCrossRefGoogle Scholar
  18. 18.
    Cheng A, Pulokas J, Suloway C, Zhu Y, Yeager M, Potter CS, Carragher B (2003) Adaptation of Leginon software for semi-automated recording of electron microscopic images of two-dimensional crystals. Microsc Microanal 9(suppl 02):1518–1519Google Scholar
  19. 19.
    Cheng A, Schweissinger D, Dawood F, Kumar NM, Yeager M (2003) Projection structure of full length connexin 43 by electron cryo-crystallography. Cell Commun Adhes 10:187–191PubMedGoogle Scholar
  20. 20.
    Cheng A, Yeager M (2007) Bootstrap resampling for voxel-wise variance analysis of three-dimensional density maps derived by image analysis of two dimensional crystals. J Struct Biol 158:19–32PubMedCrossRefGoogle Scholar
  21. 21.
    Taylor DW, Kelly DF, Cheng A, Taylor KA (2007) On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy. J Struct Biol 160:305–312PubMedCrossRefGoogle Scholar
  22. 22.
    Nakamura N, Shimizu Y, Shinkawa T, Nakata M, Bammes B, Zhang J, Chiu W (2010) Automated specimen search in cryo-TEM observation with DIFF-defocus imaging. J Electron Microsc 59:299–310CrossRefGoogle Scholar
  23. 23.
    Cheng A, Fellmann D, Pulokas J, Potter CS, Carragher B (2006) Does contamination buildup limit throughput for automated cryoEM? J Struct Biol 154:303–311PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Cell BiologyThe National Resource for Automated Molecular Microscopy, The Scripps Research InstituteLa JollaUSA

Personalised recommendations