Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection.
For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.
Wang
Liguo
L
Department of Cellular and Molecular Physiology, Yale University, 333 Cedar Street, New Haven, CT 06520, USA.
Ounjai
Puey
P
Sigworth
Fred J
FJ
D019921:Biotinylation; D002138:Calibration; D020285:Cryoelectron Microscopy / Q000379:methods / Q000592:standards*; D003460:Crystallization; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry*
D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D019809:Streptavidin