Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.
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.
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Bong-Gyoon
BG
Han
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Zoe
Z
Watson
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Hannah
H
Kang
Department of Molecular and Cell Biology and California Institute of Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
Arto
A
Pulk
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Kenneth H
KH
Downing
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.
Jamie
J
Cate
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA. Electronic address: rmglaeser@lbl.gov.
Robert M
RM
Glaeser
D001710:Biotin / Q000737:chemistry; D019921:Biotinylation / Q000379:methods*; D002244:Carbon / Q000737:chemistry; D020285:Cryoelectron Microscopy; D003460:Crystallization / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron / Q000379:methods; D012270:Ribosomes / Q000737:chemistry / Q000648:ultrastructure*; D013048:Specimen Handling; D019809:Streptavidin / Q000737:chemistry*; D013379:Substrate Specificity
D001710:Biotin; D002244:Carbon; D019809:Streptavidin
Affinity grid; Electron microscopy; Streptavidin