Traveling Through Proteomes Using 3D-EM and AFM

Nanoanalysis, July 10, 2006, ETHZAndreas Engel

Maurice E. Müller Institiute, Biozentrum

University of Basel, Switzerland

Electron Tomography

MPI of Biochemistry Max Planck Society

3D-object => set of 2D-projections3D-object => set of 2D-projections 2D-projections => 3D-2D-projections => 3D-reconstructionreconstruction

Principle of electron tomographyPrinciple of electron tomography

W. Baumeister, R. Grimm, J. Walz: Trends Cell Biol 9 (1999) 81-85W. Baumeister, R. Grimm, J. Walz: Trends Cell Biol 9 (1999) 81-85

Weighted Backprojection

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MPI of Biochemistry Max Planck Society

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Sporozoites (Sporozoites (Plasmodium bergheiPlasmodium berghei))

MPI of Biochemistry Max Planck Society

membranemembrane

microtubulesmicrotubules

ERER

dense granulesdense granules

rhoptriesrhoptries

micronemesmicronemes

polar ringspolar rings

Apical part of a sporozoite cellApical part of a sporozoite cell

Nanoanalytics of Soluble Complexes:

Scanning Transmission Electron Microscopy (STEM)

Scanning Transmission EM

200 Å

Philippe Ringler

STEM HardwareBF DF

Acceleration voltage 100 kVPressure: < 10-10 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

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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Dimitrios Fotiadis, unpublished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

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Low force

High force

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Bacteriorhodopsin: Surface Energy Landscape

6 kT

pd(r) peak positionprobability ofdomain d

Fd = -kTlnpd(r)

Scheuring et al.Eur Biophys J2002

Low force

High force

Low force High 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

AcknowledgmentsAFMDaniel Müller*Simon Scheuring* Dimitrios FotiadisPatrick Frederix

2D crystallizationHervé RémigyThomas KaufmannThomas Walz*

Protein expressionNora EiflerMyriam DuckelyPaul Werten

Peter AgreWolfgang BaumeisterYoshi FujiyoshiHelmut GrubmüllerBert deGrootKris Palczewski

STEMShirley MüllerPhilippe RinglerFrancoise Erne-Brand