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Title: Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules

Abstract

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure

Authors:
 [1];  [2];  [1];  [3];  [1];  [4];  [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
  2. Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
  3. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology and California Inst. of Quantitative Biosciense
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry; Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology and California Inst. of Quantitative Biosciense
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE; National Institutes of Health (NIH)
OSTI Identifier:
1378360
Grant/Contract Number:
AC02-05CH11231; R01-GM083039; P01-GM051487; R01-GM065050
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Structural Biology
Additional Journal Information:
Journal Volume: 195; Journal Issue: 2; Journal ID: ISSN 1047-8477
Publisher:
Elseiver
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Han, Bong-Gyoon, Watson, Zoe, Kang, Hannah, Pulk, Arto, Downing, Kenneth H., Cate, Jamie, and Glaeser, Robert M. Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules. United States: N. p., 2016. Web. doi:10.1016/j.jsb.2016.06.009.
Han, Bong-Gyoon, Watson, Zoe, Kang, Hannah, Pulk, Arto, Downing, Kenneth H., Cate, Jamie, & Glaeser, Robert M. Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules. United States. doi:10.1016/j.jsb.2016.06.009.
Han, Bong-Gyoon, Watson, Zoe, Kang, Hannah, Pulk, Arto, Downing, Kenneth H., Cate, Jamie, and Glaeser, Robert M. 2016. "Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules". United States. doi:10.1016/j.jsb.2016.06.009. https://www.osti.gov/servlets/purl/1378360.
@article{osti_1378360,
title = {Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules},
author = {Han, Bong-Gyoon and Watson, Zoe and Kang, Hannah and Pulk, Arto and Downing, Kenneth H. and Cate, Jamie and Glaeser, Robert M.},
abstractNote = {We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure},
doi = {10.1016/j.jsb.2016.06.009},
journal = {Journal of Structural Biology},
number = 2,
volume = 195,
place = {United States},
year = 2016,
month = 6
}

Journal Article:
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  • The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Since specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over themore » holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at {approx}0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25 percent-35 percent of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.« less
  • Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. Also, the quality of the Thon rings is a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 Å, while the resolution of the map reached 4.0 Å for the samemore » number of particles, when the estimated resolution of streptavidin crystal was 4 Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 Å, a marked improvement over the value of 3.9 Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Finally, streptavidin monolayer crystals appear to provide a good indication of when that is the case.« less
  • We demonstrate the application of graphene as a support for imaging individual biological molecules in transmission electron microscope (TEM). A simple procedure to produce free-standing graphene membranes has been designed. Such membranes are extremely robust and can support practically any submicrometer object. Tobacco mosaic virus has been deposited on graphene samples and observed in a TEM. High contrast has been achieved even though no staining has been applied.
  • The authors found that when they incubated SA-TG-(BCPDA){sub 150} (a conjugate of streptavidin (SA) with thyroglobulin (TG), which is labeled with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA)), with BCPDA-labeled thyroglobulin (TG-(BCPDA){sub 150}), in the presence of a suitable amount of Eu{sup 3+}, a new macromolecular complex is formed with a molecular weight of about 3 {times} 10{sup 6}. This macromolecular complex is stable and can be used as a streptavidin-based universal detection system for devising time-resolved fluorescence immunoassays. This new reagent yields an 8-26-fold improvement in the detection limits of seven different immunofluorometric assays in comparison to the reagent SA-TG-(BCPDA){submore » 150}, which they have recently described.« less