Subproject Z1:
Molecular electron microscopy
Our project aims to facilitate structural analysis of functional modules within the consortium using cryo-electron microscop (cryo-EM). Cryo-EM in combination with image processing (single particle approach) has emerged a key technology in structural biology to study the structure of large macromolecular assemblies and molecular machines. Subnanometer resolution has been achieved even with asymmetric particles such as a ribosome. However, the application of single particle techniques still requires a lot of technical expertise, and necessary steps are still far from being standardized. Project Z1 thus provides an instrumental and methodological platform, supporting collaboration partners within the SFB at different levels of cryo-EM analysis. First, we will use negative staining techniques as a routine service to evaluate the purity and homogeneity of samples provided by the members of the consortium. Next, we will collect data for initial structure determination either in negative stain or in amorphous ice, now offering automated data collection tools as a service. We will support and advice reconstruction of initial 3D models using de novo structure determination methods such as the Random Conical Tilt or Common Line approaches. In selected projects (B3 and D4), where suitable preparations have been already obtained, we will support in depth cryo-EM analysis in close collaboration with the individual partners.
In addition to supporting the members of the SFB consortium, this project also aims to develop and implement new methodology to face the two most exigent problems in cryo-EM, namely the analysis of sample heterogeneity and simplification of initial structure determination. We will continue our pioneering work on new image processing tools for multiparticle refinement, which have been proven to be essential in the analysis of intrinsic conformational variability of functional modules, which turned out to be much more distinct even within biochemically well-defined states of stable molecular machines such as the ribosome. Analysis of the sample heterogeneity will also play a crucial role for initial structure determination. Her fore, we aim to exploit molecular electron tomography (ET) in combination with subtomogram averaging as a new tool to derive initial 3D models of unknown specimen. In our case, where the macromolecules are randomly oriented on the EM-grid, a tomogram contains a number of low-resolution 3D structures from the individual complexes. Moreover, molecular ET might be the only structural technique applicable to highly dynamic protein modules with varying composition and/or high conformational flexibility. Once implemented, these techniques will made available to the SFB consortium in order to further facilitate the structural analysis of protein modules using cryo-EM.
Selected Publications (See also New Publications of the 2nd Funding Period after Reviewing CRC in 2010 and Selected other Publications of the 1st Funding Period)
Bhushan, S., Gartmann, M., Halic, M., Armache, J.-P., Jarasch, A., Mielke, T., Berninghausen, O., Wilson, D. N. & Beckmann, R. a-helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 17, 313-7 (2010)
Chen, J., Sachse, C., Xu, C., Mielke, T., Spahn, C.M.T. & Grigorieff, N. A Dose-Rate Effect in Single-Particle Electron Microscopy. J. Struc. Biol. 161, 92-100 (2008)
Connell, S. R., Topf, M., Qin, Y., Wilson, D. N., Mielke, T., Fucini, P., Nierhaus, K. H.& Spahn, C. M. T. A new tRNA-intermediate revealed on the ribosome during EF4 (LepA)-mediated back-translocation. Nat. Struct. Mol. Biol.15, 910-915 (2008)
Connnell, S. R., Takemoto, C., Wilson, D. N., Wang, H., Murayama, K., Terada, T., Shirouzu, M., Rost, M., Schüler, M., Giesebrecht, J., Dabrowski, M., Mielke, T., Fucini, P. M., Yokoyama, S. & Spahn, C. M. T. Structural Basis for Interaction of the Ribosome with the Switch Regions of GTP-bound Elongation Factors. Mol. Cell 25, 751-764 (2007)
Liu, C., Young, A. L., Starling-Windhof, A., Bracher, A., Saschenbrecker, S., Vasudeva Rao, B., Vasudeva Rao, K., Berninghausen, O., Mielke, T., Hartl, F.U., Beckmann, R. & Hayer-Hartl, M. Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature 463, 197-202 (2010)
Becker, T., Mandon, E., Bhushan, S., Jarasch, A., Armache, J.-P., Funes, S., Jossinet, F., Gumbart, J. C., Mielke, T., Schulten, K., Westhof, E., Gilmore, R. & Beckmann, R. Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 326, 1369-1373 (2009)
Munro, J. B., Sanbonmatsu, K. Y., Spahn, C. M. T. & Blanchard, S. C. Navigating the ribosome's metastable energy landscape. Trends Biochem. Sci. 34, 390-400 (2009)
Seidelt, B., Innis, C.A., Wilson, D.N., Gartmann, M., Armache, J-P., Trabuco, L.G., Becker, T., Mielke, T., Schulten, K., Steitz, T.A. & Beckmann, R. Structural insight into nascent chain-mediated translational stalling. Science 326, 1412-1415 (2009)
Schuette, J.-C., Murphy, F. V. 4th, Kelley, A. C., Giesebrecht, J., Connell, S. R., Loerke, J., Mielke, T., Zhang, W., Penczek, P.A., Ramakrishnan, V. & Spahn, C.M. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009)
Spahn, C. M. T. & Penczek, P. A. Exploring conformational modes of macromolecular assemblies by multiparticle cryo-EM. Curr. Opin. Struct. Biol. 19, 623-631 (2009)
Zhang, W., Kimmel., M., Spahn, C. M. T. & Penczek, P. A. Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis. Structure 16, 1770-1776 (2008)
Prof. Dr.
Christian Spahn
Charité - Universitätsmedizin Berlin
Campus Mitte
Institut für Medizinische Physik und Biophysik
Ziegelstrasse 5-9
Postal address:
Charitéplatz 1
10098 Berlin
+49 30 450 524 131
+49 30 450 524 931
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Dr.
Thorsten Mielke
Charité – Universitätsmedizin Berlin
Campus Mitte
Institut für Medizinische Physik und Biophysik
10117 Berlin
and
Max-Planck Institut für molekulare Genetik
Ihnestrasse 63-73
14195 Berlin
+49 30 8413 1644 / 1141
+49 30 8413 1385
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