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

Bong-Gyoon Han; Zoe Watson; Hannah Kang; Arto Pulk; Kenneth H Downing; Jamie Cate; Robert M Glaeser

doi:10.1016/j.jsb.2016.06.009

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

J Struct Biol. 2016 Aug;195(2):238-244. doi: 10.1016/j.jsb.2016.06.009. Epub 2016 Jun 15.

Authors

Bong-Gyoon Han¹, Zoe Watson², Hannah Kang¹, Arto Pulk³, Kenneth H Downing¹, Jamie Cate⁴, Robert M Glaeser⁵

Affiliations

¹ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
² Department of Chemistry, University of California, Berkeley, CA 94720, USA.
³ Department of Molecular and Cell Biology and California Institute of Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
⁴ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology and California Institute of Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
⁵ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA. Electronic address: rmglaeser@lbl.gov.

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, 2μm 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.

Keywords: Affinity grid; Electron microscopy; Streptavidin.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Biotin / chemistry
Biotinylation / methods*
Carbon / chemistry
Cryoelectron Microscopy
Crystallization / methods*
Image Processing, Computer-Assisted
Microscopy, Electron / methods
Ribosomes / chemistry
Ribosomes / ultrastructure*
Specimen Handling
Streptavidin / chemistry*
Substrate Specificity

Substances

Biotin
Carbon
Streptavidin

Abstract

Publication types

MeSH terms

Substances

Grants and funding