Traveling Through Proteomes Using 3D-EM and AFM Nanoanalysis, July 10, 2006, ETHZ Andreas Engel Maurice E. Müller Institiute, Biozentrum University of.

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Presentation transcript:

Traveling Through Proteomes Using 3D-EM and AFM Nanoanalysis, July 10, 2006, ETHZ Andreas Engel Maurice E. Müller Institiute, Biozentrum University of Basel, Switzerland

Electron Tomography

MPI of Biochemistry Max Planck Society 3D-object => set of 2D-projections 2D-projections => 3D- reconstruction Principle of electron tomography W. Baumeister, R. Grimm, J. Walz: Trends Cell Biol 9 (1999) 81-85

Weighted Backprojection

MPI of Biochemistry Max Planck Society Sporozoites (Plasmodium berghei)

MPI of Biochemistry Max Planck Society membrane microtubules ER dense granules rhoptries micronemes polar rings Apical part of a sporozoite cell

Nanoanalytics of Soluble Complexes: Scanning Transmission Electron Microscopy (STEM)

Scanning Transmission EM 200 Å Philippe Ringler

STEM Hardware BFDF Acceleration voltage 100 kV Pressure: < Torr Beam current Single electron counting PM

TMV Analysis 14 nm

Actin Collaboration with Ueli Aebi, M.E. Müller Insitute

STEM Mueller et al, J Mol Biol 99 From mass to shape

Nanoanalytics of Membrane Complexes: Atomic Force Microscopy (AFM)

Membrane Proteins exist in the Bilayer Bert de Groot & Helmut Grubmüller

Example: Bacteriorhodopsin

Cytosolic Surface of Bacteriorhodopsin Dimitrios Fotiadis, unpublished

CS of Bacteriorhodopsin: Force-induced Conformational Changes Müller et al. (1995), J. Mol. Biol. & Fotiadis et al. (2002), Micron 10 nm (A), 4 nm (B, C and D)

The Surface Dynamics of Bacteriorhodopsin Scheuring et al., European Biophysics Journal Similarity ranked images are assembled into a movie Low force High force

Bacteriorhodopsin: Surface Energy Landscape 6 kT p d (r) peak position probability of domain d F d = -kTlnp d (r) Scheuring et al. Eur Biophys J 2002 Low force High force Low forceHigh force

Unzipping Bacteriorhodopsin Oesterhelt et al. Science 2000

Conclusions Electron and atomic force microscopies offer great tools for cellular nanoanalytics Electron tomography provides entire picture of a cell STEM makes the link between mass and shape AFM is an ideal tool for assessing structure & dynamics of membrane proteins

Acknowledgments AFM Daniel Müller* Simon Scheuring* Dimitrios Fotiadis Patrick Frederix 2D crystallization Hervé Rémigy Thomas Kaufmann Thomas Walz* Protein expression Nora Eifler Myriam Duckely Paul Werten Peter Agre Wolfgang Baumeister Yoshi Fujiyoshi Helmut Grubmüller Bert deGroot Kris Palczewski STEM Shirley Müller Philippe Ringler Francoise Erne-Brand