Description of the Work Packages


WP1 (Programme for training courses) [FEI, BBK, MPIB, CSIC, LAU, EMBL, UU]

The partners of 3D-EM are organising training courses for European scientists to disseminate state-of-the-art methodology and increase the level of skills in the field of Electron Microscopy (EM). The course programme provides basic and advanced training in various aspects of EM, as electron tomography, cryo-EM and single particle analysis, both in hands-on experience and theoretical background. Detailed information on the course programme, and registration details are given on the training website (http://www.3dem-noe-training.org/). The training courses are free of charge (except for a small registration fee for some courses). In the three years of the project (March 04 - February 07), 3 workshops on CEMOVIS and 20 courses with a total of more than 300 participants were held. All courses were positively evaluated by the participants. The development of 3 DVDs for basic and advanced training in the field of electron microscopy (EM) will be completed within the fourth year. The first DVD “3D-EM in Life Science” contains the basic techniques, background knowledge and applications of Electron Microscopy in Life Science. The second “Cryo-EM of Biomolecular Complexes” and third “Electron Tomography” give a more detailed description of methodology, features and applications for experienced users. top

WP2 (Setting 3D-EM Guidelines for software development) [EMBL-EBI with all partners]

WP2 brought together software developers both in the NoE and within the world wide 3DEM community. It has reviewed tools and software practices generally used in the field of EM, identified topics requiring standardisation and allocated these developmental tasks to individual partners. This WP has been finished after the second year of the project. top


WP3 (Setting 3D-EM Guidelines for data exchange (MetaData)) [EMBL-EBI with all partners]

WP3 will continue work on the contributions to standards in the 3DEM network by using revised metadata standards that have resulted from community input that is consistent with the discipline in version 2 of the EM database and deposition system. The improved standards cover tomographic and cryo-EM experimental data and the digital images. top


WP4 (TOVIS: Tomography of vitreous sections) [LAU, UPMC, CNRS, MPI-B, CSIC, KI, EMBL]

WP4 is dedicated to the dissemination of the “CEMOVIS” (Cryo-Electron Microscopy Of VItreous Sections) technology for preparation of vitreous samples from Partner LAU to all interested institutions within and outside the NoE. Four partner institutions meanwhile established the technology. First results of tomographic analyses of vitreous sections indicate that the technology is applicable to both prokaryotic and eukaryotic cells. In combination with cryo-electron tomography, CEMOVIS will be applied to model objects such as DNA-protein complexes, ribosomes, proteasomes and Balbiani ring RNP particles. During the two years of the network, CEMOVIS was introduced at a training course and two mini-colloquium all open to public. The 3rd CEMOVIS mini-colloquium will take place in Munich from May 21st to 23rd and the 2nd CEMOVIS course in Lausanne from June 11th to 17th 2006. top


WP5 (Development of software for acquisition and processing of tomographic data) [MPI-B, LMUC, FEI, UU, EMBL, BIOZ]

The “TOM Toolbox” software package (Acquisition and Analysis for Electron Tomography) has been publicly available since May 2004. The package has meanwhile been installed in various institutions of Europe and worldwide. The TOM Toolbox software will be further extended in its capabilities towards automation of the complete workflow in tomography, addressing both high resolution and high throughput. In parallel, single particle analysis acquisition schemes will be incorporated and the software will be adapted to the new generation of electron microscopes. The addition of new partners and the establishment of the closely related new WP13 demonstrate the integrative role of this WP. In March 2006, the course “TOM software toolbox: Acquisition and analysis for electron tomography” took place for the second time. This basic course aims at giving insight into and practical experience in every step of the TOM package routines: from acquisition to 3D-reconstruction. top


WP6 (Analysis of tomographic data) [UPMC, CNRS, ICSR, CSIC, EMBL]

This project focuses on improving the sorting and merging of 3D reconstruction volumes originating from tomographic data sets. For this purpose, new algorithms using self-organizing maps (SOM) based on neural networks are adapted for the classification of 3D data. Tomography of subcellular structures and macromolecular assemblies will be continued and improvements respectively developments will be made regarding the 3D reconstruction of heterogeneous samples, the segmentation and denoising of 3D maps, and user friendly interfaces. Moreover, new emphasis will be given to the development of alignment algorithms for tomographic series, for determination and correction of the CTF (contrast transfer function) to improve the resolution of 3D data sets. A complementary approach, combining icosahedral particle and conventional reconstruction methods, especially of viruses within cellular environments, will be carried out. Cells infected by viruses will be used for this purpose. Synthetic as well as experimental data sets will be used for testing noise-fitting, data restoration and segmentation algorithms. In addition the methods will be applied to the 3D study of normal and pathological centrosomes implicated in breast cancer. top


WP7 (Data processing in electron crystallography) [BIOZ, CSIC, MPI-F, KI]

The single effort in this Network Area is WP7 "Data processing in electron crystallography". Its goal is the implementation of a comprehensive electron crystallography image processing pipeline within the context of the IPLT programme suite (Image processing library & toolbox, http://www.iplt.org/), in combination with other high-level python modules for Xmipp and MRC. top

WP8 (Electron tomography of individual macromolecules) [KI]

The Karolinska Institute group has recently managed to study individual macromolecules with ET through the introduction of the iterative refinement procedure COMET (JSB 117, 173-188, 1996). This procedure allows for reproducible 3D electron tomography (ET) reconstructions at about 2 nm resolution of native proteins in flash-frozen buffer, or about 2-3 nm resolution on lightly stained frozen hydrated (PVA embedded) sections of cells and tissues. Currently proteins of the size 20 kD and up have been studied. The programs defining the COMET software will be made available to the members of the NoE as executables under non-commercial licenses, and the application of the technology will be stimulated and supported by specific courses and direct interactions between the individual laboratories. Proteins with well-known X-ray structure will be used to test and to improve the newly developed ET method. In this context we will focus on an increase in resolution by using liquid helium instead of liquid nitrogen as well as on the further development of software to find and extracts objects of proper size. top

WP9 (Optimised strategies for single particle reconstruction) [BBK, UPMC, UOX, Charite]

Standard image processing strategies for single-particle analysis are based on the assumption that the macromolecular complexes are homogeneous. Therefore these techniques are particularly successful for samples with little conformational variability However, most samples in practice exhibit significant heterogeneity. In order to cope with the associated problems, image processing strategies will be developed for particles exhibiting substantial variation in conformation and/or ligand occupancy. The procedures will form the basis for quantitative statistical analysis of conformational variability and sample heterogeneity based on randomized sub-sampling techniques. New protocols will be tested on both simulated data and on several real data sets of asymmetric and symmetric particles. As a prerequisite for a routine application of the new protocols, efforts will be made to automate image processing steps which are so far optimized interactively. A smaller objective on the side of WP9 is to help molecular interpretation of high resolution cryoEM 3D maps, using folding and molecular modelling approaches. top


WP10 (The development of control and measurement software for use at the microscope level) [Imperial, LUMC]

The main objective of this workpackage is to identify and examine key issues associated with electron microscopical data collection that limit the overall resolution attainable with the single-particle cryo-EM technique. To this end, we are currently pursuing two main lines of research: a) strategies for improved TEM alignment and improved CTF correction to enable us to get the most out of the data collected, and b) the use of controlled-astigmatism in collecting data to obviate the need for collecting micrographs at a range of different defoci. top


WP11 (Towards a unified resolution measure for 3D EM) [Imperial, CNRC]

Cross correlation coefficients (CCCs) are used routinely in many 2D and 3D procedures in single-particle cryo-EM, such as Multi Reference Alignments (MRA), Projection Matching, and “fitting” of PDB co-ordinates or other 3D structures in low-resolution 3D EM maps. We are defining new information-based similarity measures which will be used in all relevant places, such as the MRA procedures in IMAGIC (in collaboration with Image Science GmbH, Berlin). One of the applications is to sort out mixtures of conformational states that are often present in an ensemble of single molecules in solution. We also wish to incorporate 3D versions of the better correlation measures into “fitting” procedures used for interpreting the 3D cryo-EM reconstructions. The new software developments required a new “in core” 3D subroutine library (Image Science). This new library has been used for a first version of an “automasking” programme which can find areas of interest in a large 3D volume. top


WP12 (Project management) [BIOZ, MPI-B, FEI, CSIC, KI, BBK]

The management and coordination of 3D-EM is a shared responsibility between the partners BIOZ and MPI-B. The Coordinators office in Basel oversees project coordination and administration along with the communication with the European Commission. The project office at the MPI-B is responsible for scientific management, public relations and dissemination (incl. website) and the management of the 3D-EM Fellowship Programme. top


WP13 (Development and implementation of correlative light microscopy and cryo electron-tomography) [LUMC, MPI-B, FEI, UU, CNRS, ICSR]

WP 13 is dedicated to the development and implementation of techniques for correlative light microscopy and cryo electron-tomography. Fluorescence microscopic techniques and fluorescent labelling protocols will be evaluated and further developed as tools to facilitate the identification of regions of interest for subsequent high-resolution analysis by cryo electron tomography. In parallel, a software module for the TOM Toolbox will be written in collaboration with MPIB to correlate light microscopic with electron microscopic images. top