Dierk Niessing (STB) •Group Intracellular Transport & RNA Biology •X-ray crystallography platform X-ray crystallographic and functional studies on gene regulation by mRNA localization Why X-ray crystallography at HMGU? What do we use it for? Depositions of structures in public repository PDB Source: PDB data base 2012 Comparison of the major structure techniques Size: (kDa) 0 25 50 Domains & protein fragments NMR 100 200 400 Larger proteins & protein complexes 800 … Ribosomes, viruses etc Cryo-electron microscopy X-ray crystallography Principles of protein crystallization Electron density calculation Crystal size: 0.01-0.5 mm Model building X-ray Crystallography platform at STB Setup of crystallization screensin 96 well format TTP-Mosquito robot for 96- (384-) well plates Automatic plate sealer 20°C or 4°C incubation SBGrid software package @ HMGU X-ray source Leica stereo-microscopes Access to synchrotron beamlines for high-quality data collection ESRF in Grenoble, France: monthly access shared with LMU, TUM, and MPI DESY (PETRA III) in Hamburg: Beamline P11: station for data collection 1/3 of time for Helmholtz Association 1/3 of time for Max-Planck Society 1/3 of time for outside scientists A look at PETRA III and the new P11 beamline Why X-ray crystallography at HMGU? What do we use it for? mRNA localization and local translation is essential for asymmetric cellular events Xenopus eggs Rat neurons Drosophila embryos Nucleolus mRNA localization in yeast Heuck et al. & Niessing PNAS (2007) She2p Nucleolar transition Myo4p cargo domain Complex reconstitution Müller et al. & Niessing RNA (2009) Niessing et al. & Burley Cell (2004) Niedner, et al. & Niessing PNAS (2013) Heuck et al. & Niessing J. Cell Biol. (2010) Müller, Heym et al. & Niessing PLoS Biol. (2011) X-ray structures Yeast Bim1p Yeast Myo4p Human Myo5a Heuck et al. J CellBiol 2010 Hüls et al. JBC 2012 Velvarska & Niessing PLoS One 2013 Yeast She2p Niessing et al. Cell 2004 Human Myo5b Velvarska & Niessing PLoS One 2013 Prokaryotic PurPur- alpha Drosophila PurPur- alpha II- II Graebsch et al. PNAS 2009 Graebsch et al. PLoS ONE 2010 Strategy for single molecule motility assay GFP GFP GFP GFP Heym, Zimmermann et al. & Niessing J. Cell Biol. (2013) Single-particle motility assay Green: GFP-labeled particle Red: Actin filaments Niessing lab Franziska Edelmann Simone Heberlein Robert Janowski Annika Niedner Vera Roman Jana Tretter Janine Weber Former members: Almut Graebsch Daniela Hüls Alexander Heuck Roland Heym Sigrun Jaklin Marisa Müller Janina Pfaff Stephane Roche Hana Velvarska Heym, Zimmermann et al. & Niessing J. Cell Biol. (2013) Collaborations Klaus Richter Johannes Buchner (TUM) Janosch Hennig Michael Sattler (HMGU/TUM) Gunter Meister (Uni Regensburg) David Kovar (Chicago University) Zeynep Oekten (TUM) Henning Urlaub (Max Planck - Göttingen) Ralf-Peter Jansen (Tübingen University) Jean-Christophe Paillart Patrick Cramer (Munich University) (University Strassburg) Marilyn Munson (University Massachussets) Aubry Miller (DKFZ) Klaus Förstemann (LMU) DFG FOR855 / SFB646 Boehringer Ingelheim Fonds Wilhelm-Sander Stiftung