Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr.
We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60-kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween-80 display hexagonally close-packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge-freezing, and (ii) frozen-hydrated sections obtained from cells frozen under high pressure. The frozen-hydrated sections were generated by sectioning at -150 degrees C using a diamond knife with a 25 degrees knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen-hydrated sections and the arrangements found in negatively stained membrane extracts and plunge-frozen cells, highlighting the potential of using frozen-hydrated sections for the study of macromolecular assemblies within cells under near-native conditions.
Zhang
P
P
Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, MD 20817, USA.
Bos
E
E
Heymann
J
J
Gnaegi
H
H
Kessel
M
M
Peters
P J
PJ
Subramaniam
S
S
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000648:ultrastructure*; D015925:Cryopreservation; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000648:ultrastructure*; D008854:Microscopy, Electron; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000648:ultrastructure*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C057635:Tsr protein, Bacteria