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An Improved Holey Carbon Film for Cryo-Electron Microscopy

Published online by Cambridge University Press:  28 September 2007

Joel Quispe ,
John Damiano ,
Stephen E. Mick ,
David P. Nackashi ,
Denis Fellmann ,
Teddy G. Ajero ,
Bridget Carragher  and
Clinton S. Potter
Joel Quispe
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
John Damiano
Affiliation:
Protochips, Inc., Raleigh, NC 27603, USA
Stephen E. Mick
Affiliation:
Protochips, Inc., Raleigh, NC 27603, USA
David P. Nackashi
Affiliation:
Protochips, Inc., Raleigh, NC 27603, USA
Denis Fellmann
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
Teddy G. Ajero
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
Bridget Carragher
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
Clinton S. Potter
Affiliation:
The National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
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Abstract

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

Keywords

TEMcryo-electron microscopyholey carbon filmsautomation
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 13 , Issue 5 , October 2007 , pp. 365 - 371
Copyright
© 2007 Microscopy Society of America

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References

REFERENCES

Downing, K.H. (2003). Support films with uniform hole size. Microsc Today 11, 54.Google Scholar
Dubochet, J., Adrian, M., Chang, J.J., Homo, J.C., Lepault, J., McDowall, A.W. & Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21, 129–228.Google Scholar
Ermantraut, E., Wohlfart, K. & Tichelaar, W. (1998). Perforated support foils with re-defined hole size, shape and arrangement. Ultramicroscopy 74, 75–81.Google Scholar
Henderson, R. (1995). The potential and limitations of neutrons, electrons, and X-rays for atomic resolution microscopy of unstained biological macromolecules. Q Rev Biophys 28, 171–193.Google Scholar
Murray, J. (1987). Preparation of holey carbon films suitable for cryo-electron microscopy. J Electron Microsc Tech 5, 285–290.Google Scholar
Quispe, J., Banez, R., Carragher, B. & Potter, C.S. (2004). Improving automation for cryo-EM specimen preparation. Microsc Microanal 10(Suppl. S02), 1508–1509.Google Scholar
Stagg, S.M., Lander, G., Pulokas, J., Fellmann, D., Cheng, A., Quispe, J.D., Mallick, S.P., Avila, R.M., Carragher, B. & Potter, C.S. (2006). Automated cryoEM data acquisition and analysis of 284,742 particles of GroEL. J Struct Biol 155, 470–481.Google Scholar
Suloway, C., Pulokas, J., Fellmann, D., Cheng, A., Guerra, F., Quispe, J., Stagg, S., Potter, C.S. & Carragher, B. (2005). Automated molecular microscopy: The new Leginon system. J Struct Biol 151, 41–60.Google Scholar
Taylor, K.A. & Glaeser, R.M. (1974). Electron diffraction of frozen, hydrated protein crystals. Science 186, 1036–1037.Google Scholar