1.1 Identifying relevant folding units in large molecular complexes

- Construction of cell lines stably expressing a GFP target protein (BRCA1). Creation of a Cyan Fluorescent (CFP) tagged library of human proteins.

- Development and implementation of FRET assay on a FACS machine. Screening of CFP tagged protein library. Cloning and sequencing of proteins detected in the screen.

- Determining protein domains for the most interesting interacting proteins.

- Preparation of appropriate constructs for structural studies of interacting proteins found in the domain FRET screen.

1.2 Characterizing protein structure and dynamics in crowded solutions

- a system for measuring protein diffusion rates in nanoliter volumes;
- characterizing effects of crowding on weak protein complex with NMR.

- feasibility study of pre-screening protein crystallization trials by EM;
- a generic system for predicting the likelihood of protein crystallization conditions based on diffusion rates in nanoliter volumes.

1.3 Characterizing insoluble proteins and protein complexes by solid state NMR

- To design and perform MAS NMR experiments to refine the structural information.

- Incorporate current structural knowledge of Alzheimer plaques in virtual studio;- To perform assays of DNA-Histone interactions to resolve the differences between the chromatin structures in healthy and sick cells.

1.4 Simultaneous refinement X-ray structures & reciprocal space density modification

- a computer program for joint refinement of multiple crystal structures;
- a maximum likelihood, multivariate formulation of phase restraints due to prior low-resolution information.

- computer program for multivariate, reciprocal space density modification.

1.5 Integrating single protein studies with low resolution studies of assemblies & single particle analysis

- the flash option for UV, electrostatic triggered experiments and the glove box for cryo fixation of cells and tissues have been developed
- Leginon transferred into a professional software product for single particle data acquisition, including database management and 3D reconstruction;
high throughput data collection is working, allowing the collection of up to 10⁵ individual views of single particles per 24 h;
- picking particles fully automated;
- parallel software architecture for data processing;
- new computational approaches to deal with phase contrast and prior information.

- The pathway from sample preparation to sample analysis is fully automated;
-Leginon is a generic tool for characerising the functioning of molecular machines;
- high throughput data collection is the routine mode of operation;
- stand-alone computer program for routine structure determination, defocus tolerant and including solvent flattening and prior high resolution information.

1.6 Visualizing cell – and membrane surfaces

- development of proper immobilization techniques of native proteins and membranes;
- development of novel techniques to specifically bind biomolecules to AFM tips.

- Atomic-force microscopy (AFM) as a versatile tool for obtaining sub-molecular structures of biomolecules and biomolecular aggregates on surfaces of life cells;
- novel contrast mechanisms for scanning-probe microscopy (SPM) to directly identify biomolecules embedded in complex environments;

2.1 Tomography of single cells

- proof of principle of ultra-small gold labeled llama antibody localization of proteins;
- cryo-tomography with dual-axis cryo-holder is feasible and produces high resolution 3D structural information;
- formulation of new principles in 3D restoration by electron-tomography.

- 3D electron tomography is a generic, low threshold approach in nm resolution cellular imaging and protein localization.

2.2 Single molecule detection in live cells

- novel labeling opportunities for applications in cell biology evaluated;
- ultrastructural mapping of membrane domains in cancer pathways .

- platform for fluorescence imaging, resolution better than 50 nm;
- platform for multi-color, multi-parameter single-molecule imaging;

3.1 Integration of parameters of cellular processes on all relevant time and length scales

- connection of ultrastructural mapping with global cellular signaling on the timescale of the relevant signaling processes;
- integrate fluorescence-correlation microscopy in 4D-microscopy;
- tools to follow chromatin modeling and gene regulation in living cells.

- a platform of optical microscopies able to follow molecular interactions in the time-frame of 100 ms to 1 hour and at a resolution of 1-5 or 50-200 nm;- initial steps of signaling cascades can be followed at cellular membranes.

3.2 Extending NSOM and multimodal imaging

- proof of principle of thin-film array technology;
- establish parameters for observing dynamic behavior of cells by NSOM.

- robust platform for multimodal microscopy, combining AFM, NSOM and confocal microscopy for studying cellular and ultrastructural dynamics.

3.3 Imaging cells inside organisms

- a system for recording MRM movies with a time frame less than a second.

- a system for recording MRM images with a sub micron resolution.

4 Databases, modeling and visualization

- Prototype of database infrastructure is available, together with a number of tools for the visualization of raw data, for image search and for connection of different modalities at the same scale.
- A reciprocal-space visualization system for raw crystallographic data.
- visualization program for 3D molecular docking studies.

- Prototype of Common Visualization Platform is available, linking the different modalities, provides visualization, query and browsing facilities.