Low-energy-loss electron microscopy of doxorubicin in human breast cancer MCF-7 cells: localization by color.

The distribution of the anti-cancer drug doxorubicin (DOX) in human breast cancer MCF-7 cells was imaged directly by low-energy-loss electron microscopy (EM) without specific antibodies or heavy metal stains, using only the electron-induced molecular orbital excitation of the drug. Cells treated with DOX were examined live by confocal fluorescence microscopy and as very thin sections in an electron microscope equipped with an electron energy filter having an energy resolution of 1 eV. The distribution of DOX obtained by EM from pairs of images at energy losses of 3+/-1 eV and 10+/-1 eV agreed with fluorescence microscope observations, but provided much more detail, easily distinguishing localization between nuclear membrane and perimembrane compartments and between vacuolated nucleoli and perinucleolar chromatin.

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy.

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP.

3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA transposition.

Mu DNA transposition proceeds through a series of higher-order nucleoprotein complexes called transpososomes. The structural core of the transpososome is a tetramer of the transposase, Mu A, bound to the two transposon ends. High-resolution structural analysis of the intact transposase and the transpososome has not been successful to date.

Structure-based de novo design of ligands using a three-dimensional model of the insulin receptor.

For the first time, a three-dimensional model of the insulin receptor is used in the de novo design of novel ligands that potentially mimic interactions of insulin at its receptor. Compound 4 competed with insulin as seen in autophosphorylation assays and inhibited up to 68% of IR autophosphorylation at 300 microM of 4 in 3T3IR cells induced by 1 nM insulin. This model provides a basis for the design of potent insulin receptor ligands.

Freestanding lipid bilayers as substrates for electron cryomicroscopy of integral membrane proteins.

Phospholipid bilayers, 40 A thick, were generated as electron microscope substrates by submerging copper grids overlaid with holey plastic through a lipid monolayer on a water surface. Previously formed proteoliposomes containing single-particle membrane proteins in their bilayers were then fused into the newly formed bilayer substrate. To demonstrate this methodology, multi-drug resistance protein P-glycoprotein was incorporated into these bilayers and imaged by fixed beam microscopy and scanning transmission electron microscopy.