New common technology platforms

Objective 1. Establish a high-end cryo-electron microscopic facility at the LMU (2007)

The Beckmann lab will offer to CIPSM expertise and one of the most advanced facilities world-wide for cryo-EM and single particle analysis. The facility is currently set up and will include a 100 kV routine EM microscope Morgagni (FEI) for screening, a 120 kV routine cryo-EM microscope Spirit (FEI) including 2kx2k CCD camera for screening, initial 3D reconstruction, tomography, and low to medium resolution 3D reconstruction, a 300 kV FEG high-end cryo-EM microscope Titan-Bio (FEI) including 4kx4k CCD camera, for most advanced automated cryo-EM at the highest resolution, an automated sample making device (Vitrobot), a high-end scanner (Heidelberg) for high resolution micrograph scanning, and a computer cluster (64-128 CPU 64-bit Opteron system) for high performance image processing and 3D reconstruction, including graphical workstations for visualization, model building, and interpretation. The position of a cryo-EM scientist is provided by LMU-Innovative A. Due to a high number of collaborative projects, an additional technician position for the cryo-EM facility will be provided by CIPSM.

 

Objective 2. Establish an advanced bioanalytics platform (2007)

Present instrumentation in advanced bioanalytics will be pooled and extended by new technologies into a common platform for CIPSM. Currently available techniques include surface plasmon resonance and fluorescence polarisation (Jung, Conti), Edman sequencing (Cramer), and fluorimetry, UV/Vis spectroscopy, and CD (Carell; Cramer, Hopfner). A scientist position is established in the Jung lab to supervise and run several bioanalytics instruments. Work has been initiated to establish a facility to test protein-protein interactions and map interacting domains in a high-throughput manner based on surface plasmon resonance (Conti), to map structured crystallizable protein domains by NMR (Sattler) and by deuterium labelling and mass spectroscopy (Hopfner).

 

Objective 3. Establish a HTP crystallization and visualization facility (2007)

Access to a robotic platform that automates finding of good crystallization conditions in short time frames with a minimum of sample is of paramount importance, particularly for tackling large biological complexes. To this end, a High-Throughput Crystallization and Visualization (HTCV) platform will be established under the supervision of the Conti lab that is precise, efficient, robust, scalable, and accessable to the CIPSM members. The HTCV hardware will include a liquid handling robot for the automatic set up of crystallization solutions and for filling the reservoirs of 96-well plates, a nanodrop robot to dispense the protein sample in the prefilled plates, and visualization robots at 18 C and at 4 C for storage and automatic monitoring of crystal growth. The hardware will be installed in a 60 m2 space in the Dept. of E. Conti at MPI. The participants of the Cluster will book the crystallization screenings of choice on a web-based system and will handle the protein sample to a technician dedicated to operating the robots. Crystallization plates will be identified by barcodes for experiment tracking. This set-up has worked efficiently at EMBL for the past two years and the know-how will be transferred to MPI in 2007. The crystallization trays will be placed in one of the two robotic systems for storage (at 18 C or at 4 C) and images will be collected automatically at regular intervals with high quality microscopes. The users will view images of their experiments remotely via a web-based browsing tool. Part of the facility is funded by start-up funds from E. Conti MPI appointment. Specifically, Conti will provide a dedicated half-time technician position and will fund the liquid handling robot (Tecan Miniprep, 50 kEu), two visualization robots with storage for 150 trays each (Tecan Freedom EVO 150 with Kryos imaging stations, 250 kEu) and the nanodrop robot (Mosquito, 90 kEu). Based on the experience in running a similar facility at EMBL and the number of interested groups in the Cluster the expected request is of 400/500 plates per month. One crystallization plate consists of 96 conditions, is performed with 20 l of protein and costs 15 Euro in terms of reagents (trays, solutions and tips). To make this high-end platform available to CIPSM members, a half-time technician position (matching the one already provided by MPI) will be provided by CIPSM. Consumables must be paid for by the users. To increase the capacity of the visualization robots an additional carousels with storage for additional 280 plates (55 kEu) will also be provided.

 

Objective 5. Establish a yeast functional genomics platform (2007)

The platform that is currently available in the Jentsch lab includes the following yeast libraries and strains in a 384-well format: (A) A two-hybrid (Y2H) library of all yeast ORFs. (B) A knock-out library (KO) of all non-essential yeast genes. (C) A library of all yeast ORFs cloned as GFP fusions. (D) A library of all yeast ORFs cloned with a TAP-tag. The libraries of (A) and (B) can be used for robot-based screens to identify interacting proteins. The screens are based on mating of all strains of the library with the cells that express, or are mutant in, a gene of interest, respectively. Y2H screens allow the identification of physically interacting partners, and one screen takes about 2 weeks. The KO-library is useful to screen for (1) specific phenotypes of the mutants (e.g. MMS sensitivity) or (2) to screen for synthetic lethal (SL)/growth phenotypes of mutants in combination with a deletion mutant of the library. This type of screen can be done in four weeks. Also the GFP library (C) can be used to screen for altered intracellular localizations of GFP-tagged proteins in a mutant background of interest. In addition, all the aforementioned libraries, plus the library (D) are extremely useful just for the reason to obtain a particular mutant or fusion protein for functional studies. All four libraries are amplified and stored in the Jentsch lab and are ready to use. The libraries have been tested and the computer programs for the robot-based screens have been optimized. The robot-based setup (Beckman, Biomek FX) is currently only used by the Jentsch department, and about 1–2 (parallel) screens run each week. By optimizing the schedules for the robot, it will be possible to run up to 4 parallel screens per week. To make the platform available for CIPSM groups, it will be necessary to expand it by half a technician position for making the media, the plates, to organize and maintain the libraries, and to help with the screens (to be matched by MPI). The screens will however be performed by the users after instructions. In addition, a lockable double-door freezer will be purchased for storing the libraries separate from the materials of the Jentsch group.

 

Objective 6. Establish course in advanced structural network biology (2008)

Members of this research area will establish an advanced course in structural networks biology for graduate students and postdoctoral researchers. The idea of the course is to educate a new generation of scientists who will be able to apply both structural biology and genome-wide functional techniques to a given biological system. At present, most graduating PhD students and postdocs are either educated to work as structural biologists, yeast geneticists, cell biologists, or mass spectrometry experts. The idea of the course is to provide additional education beyond the own field of expertise and create an awareness and an understanding of related areas. The course will include lectures, technological demonstrations and some hands-on experiments. It will be offered once a year as a rotation course where each member of the research area is responsible for 1–2 days and will concentrate on their own special expertise. The course will be open to CIPSM member labs.

 

Objective 8. Establish an advanced biological NMR platform (2007–2008)

The Kessler lab has a continued interest in developing and implementing new NMR techniques for studies of higher molecular weight systems, to improve the quality of NMR-based structure determination, and to characterize conformational dynamics in proteins and protein-ligand complexes. For example, they are developing an approach based on residual dipolar couplings and relaxation enhancements by spin labelling to define the quaternary structure of macromolecular complexes in solution. An additional strategy combines orientational restraints from RDCs with “shape” information from small angle scattering data (SAXS/SANS). In this context, the neutron reactor in Garching will be available to provide SANS data for structure determination of macromolecular complexes in solution, to provide complementary information to NMR and X-ray crystallography. A number of collaborations already exist with complementary structural biology and biophysical methods (X-ray crystallography, SAXS, SANS, EPR, EM) for multidisciplinary studies of proteins and protein-ligand complexes. These collaborations exploit and combine the respective advantages of the different methods. For example, structure determination of larger proteins using X-ray crystallography can be combined with the use of NMR for binding site mapping and for the analysis of conformational dynamics. For several CIPSM members it will be important to establish an advanced NMR facility for the identification of small molecules inhibitors of potential drug targets, to study the modulation of protein function by small molecules and/or metabolites, and to provide help with isotope labelling for NMR structure determination. These efforts can provide a molecular link for the analysis of protein networks towards systems biology. To operate such a NMR facility, a scientist position is required.

 

Objective 9. Take over 30% of the Swiss Light Source (SLS) beamline PX2 (2009)

The Swiss Light Source (SLS) is a state-of-the-art third-generation synchrotron source located only 4 hours driving from Munich. Many CIPSM groups require synchrotron beamtime for crystal structure determinations. Currently the Max-Planck society holds a 50% share of the high-brilliance protein crystallography beamline PX2 at SLS. In 2009, the MPG will however invest heavily at Petra III in Hamburg and can therefore make most of the beamtime at SLS available to CIPSM. Here it is proposed that CIPSM covers 250,000 € running costs for the PX2 beamline from central funds, corresponding to about 30% of the beamtime, leaving 20% to MPG members. This would provide very valuable ensured beamtime access to CIPSM members at a short notice, as required for highly competitive structural biology projects.

 

Objective 10. Establish mail-in crystallography at the SLS beamline PX2 (2009)

 

Objective 11. Actively participate in the foundation of a joint “Munich Center for Molecular Systems Biology” (MPG, LMU, TUM)

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