traveling through proteomes using 3d-em and afm nanoanalysis, july 10, 2006, ethz andreas engel...
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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Dimitrios Fotiadis, unpublished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
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