Final Report Summary - HT3DEM (High-throughput three-dimensional Electron Microscopy)
The general aim of HT3DEM was to enhance the European leadership in three-dimensional electron microscopy (3D EM). Specifically, HT3DEM developed an automated platform to enable high throughput screening and analysis of native protein complexes and two-dimensional (2D) membrane protein crystals by EM. For this purpose, HT-3DEM brought together European experts in EM, 2D crystallisation, machine vision and robotics with leading manufacturers of electron microscopes and micro systems technologies. The innovative technology platform includes:
(i) the preparation of 2D membrane protein crystals;
(ii) automated EM-sample grid preparation and transfer to the EM;
(iii) automated image acquisition and sample analysis by EM; and
(iv) management of all data produced in the pipeline.
Each of these modules represents a substantial advance of the state of the art, and greatly enhances the throughput in 3D EM. With the successful realisation of the high throughput chain, the reduction of processing time and cost for individual samples opens new opportunities of applications, among others, in health care (screening of patient materials for infective agents) and pharmaceutical industry (acceleration of rational drug design, improving drug formulations).
After extensive testing and crystallisation of several membrane proteins the 2DX robot has reached high operational reliability and robustness over days. Purified protein is mixed in a 96-wellplate with the desired variation of buffers, salts, lipids, and detergents on the easily programmable ternary mixture robot. The detergent concentration is measured in the in-house developed so called Drop-Box. The crystallisation method used with the present 2D crystallisation robot is based on the neutralisation of detergent by the controlled addition of cyclodextrins. Features like temperature control of the sample plate, light scattering device for monitoring sample aggregation in the wells, liquid level control and a user-friendly graphic interface allow maximum degrees of variability in the crystallisation process.
Based on extensive tests a redesigned beta-version of the database has been completed. Users can design crystallisation experiments, assign a unique identifier to each sample, upload a crystallisation program in the 2DX robot and link EM data (magnification, micrographs etc.) to their crystallisation screens. Search and statistical analysis engines are being developed with the growing collection of representative data sets.
After four years of intensive development within the frame of the Sixth Framework Programme (FP6) project, we have achieved a fully operational high throughput chain for the 2D-crystallisation of membrane proteins. Extensive applications are under way and undoubtedly will lead to further improvements. However, with the first and most important step demonstrating not only the rationalised high throughput crystallisation process but more importantly also the adequate sample preparation and image analysis process, done, the way has been opened for the development of further applications in health care (e.g. screening of patient materials for infective agents) and pharmaceutical industry (e.g. acceleration of rational drug design, improving drug formulations).
(i) the preparation of 2D membrane protein crystals;
(ii) automated EM-sample grid preparation and transfer to the EM;
(iii) automated image acquisition and sample analysis by EM; and
(iv) management of all data produced in the pipeline.
Each of these modules represents a substantial advance of the state of the art, and greatly enhances the throughput in 3D EM. With the successful realisation of the high throughput chain, the reduction of processing time and cost for individual samples opens new opportunities of applications, among others, in health care (screening of patient materials for infective agents) and pharmaceutical industry (acceleration of rational drug design, improving drug formulations).
After extensive testing and crystallisation of several membrane proteins the 2DX robot has reached high operational reliability and robustness over days. Purified protein is mixed in a 96-wellplate with the desired variation of buffers, salts, lipids, and detergents on the easily programmable ternary mixture robot. The detergent concentration is measured in the in-house developed so called Drop-Box. The crystallisation method used with the present 2D crystallisation robot is based on the neutralisation of detergent by the controlled addition of cyclodextrins. Features like temperature control of the sample plate, light scattering device for monitoring sample aggregation in the wells, liquid level control and a user-friendly graphic interface allow maximum degrees of variability in the crystallisation process.
Based on extensive tests a redesigned beta-version of the database has been completed. Users can design crystallisation experiments, assign a unique identifier to each sample, upload a crystallisation program in the 2DX robot and link EM data (magnification, micrographs etc.) to their crystallisation screens. Search and statistical analysis engines are being developed with the growing collection of representative data sets.
After four years of intensive development within the frame of the Sixth Framework Programme (FP6) project, we have achieved a fully operational high throughput chain for the 2D-crystallisation of membrane proteins. Extensive applications are under way and undoubtedly will lead to further improvements. However, with the first and most important step demonstrating not only the rationalised high throughput crystallisation process but more importantly also the adequate sample preparation and image analysis process, done, the way has been opened for the development of further applications in health care (e.g. screening of patient materials for infective agents) and pharmaceutical industry (e.g. acceleration of rational drug design, improving drug formulations).
