TY - JOUR
T1 - Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr
AU - Zhang, P.
AU - Bos, E.
AU - Heymann, J.
AU - Gnaegi, H.
AU - Kessel, M.
AU - Peters, P. J.
AU - Subramaniam, Sriram
PY - 2004/10
Y1 - 2004/10
N2 - We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells over-producing 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°C using a diamond knife with a 25° 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.
AB - We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells over-producing 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°C using a diamond knife with a 25° 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.
KW - Chemotaxis receptor
KW - Cryoelectron microscopy
KW - Diamond knife
KW - Frozen-hydrated section
KW - High-pressure freezing
KW - Signal transduction
UR - http://www.scopus.com/inward/record.url?scp=4744346378&partnerID=8YFLogxK
U2 - 10.1111/j.0022-2720.2004.01395.x
DO - 10.1111/j.0022-2720.2004.01395.x
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C2 - 15369487
AN - SCOPUS:4744346378
SN - 0022-2720
VL - 216
SP - 76
EP - 83
JO - Journal of Microscopy
JF - Journal of Microscopy
IS - 1
ER -