Transcontinental EM Initiative for Membrane Protein Structure:


3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy

Transcontinental EM Initiative for Membrane Protein Structure

In this paper, two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes.

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Automated Staining Robots

Transcontinental EM Initiative for Membrane Protein Structure

We describe here the use of a commercially available magnetic plate and liquid-handling robot for the automated staining of 96 TEM grids.

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Automatic TEM image acquisition and analysis of 2D-crystallization trials

Transcontinental EM Initiative for Membrane Protein Structure

Animated-TEM software for automatic screening of 2D crystallization trials has been implemented on a JEOL1230 electron microscope running the Leginon program for controlling the electron microscope. This integrated system offers a way to robotically evaluate crystallization screens by electron microscopy.

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Cyclodextrin robot

Transcontinental EM Initiative for Membrane Protein Structure

We describe here the use of a cyclodextrin robot, used for 2D crystallization of membrane proteins. Cyclodextrin neutralizes detergent molecules with accurate detergent-dependent stoichiometry. The kinetics of the reconstitution processes is precisely controlled by quantitative addition of a cyclodextrin solution to the reaction mixture containing solubilized proteins and lipids.

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Designing a sparse-matrix screen for 2D crystallization of membrane proteins

Transcontinental EM Initiative for Membrane Protein Structure

For X-ray crystallography, sparse-matrix screens are well established for both soluble and membrane protein targets. In order to extend this approach to two-dimensional crystallization of membrane proteins, we have analyzed previous successful experiments in order to uncover relationships between relevant parameters.

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