TOM TOM A toolbox toolbox for for Cryo Cryo- -Electron - - PowerPoint PPT Presentation

TOM TOM A A toolbox toolbox for for Cryo Cryo-

• Electron

Electron Tomography and Single Particle Tomography and Single Particle Analysis Analysis

Stefan Bohn Stefan Bohn

Max Planck Institute

• f Biochemistry

Martinsried, Germany

MAX PLANCK SOCIETY

15th of November 2007

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... ...more more than than 200 200 programs programs ...more than 150 ...more than 150 labs use it labs use it worldwide worldwide Aquisition Aquisition Alignment Alignment Analysis Analysis Reconstruction Reconstruction

www.biochem.mpg.de/tom www.biochem.mpg.de/tom

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Protein Protein breakdown breakdown by by the the ubiquitin ubiquitin-

• proteasome

proteasome pathway pathway

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11nm 15nm

• The proteolytic core complex
• Conserved architecture from

archaea to humans

20S core 20S core

α α β β β β α α

• A multi-subunit (>32) complex
• 2.5 MDa
• Degrades ubiquitinated

substrates in an ATP-dependent manner

19S cap 19S cap 19S cap 19S cap

26S Proteasome 20S Proteasome

Co Co-

• star: the 26S and 20S proteasome

star: the 26S and 20S proteasome

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10 nm

The 20S proteasome of The 20S proteasome of T. acidophilum

• T. acidophilum

top top view view side side view view

10 nm

Loewe et. al.,Science Loewe et. al.,Science 268 268 (1995) (1995)

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Gold Gold labelled labelled Ubiquitin Ubiquitin-

• aldehyde

aldehyde binds binds 26S 26S specifically specifically

Hölzl et al., 2000

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Integration of tomography and SPA in TOM Integration of tomography and SPA in TOM

Semi-Automated Acquisition Projection Alignment Reconstruction Volume Visualization Particle picking Particle Alignment Classification Tomography Single Particle Analysis 3D reconstruction 3D reconstruction

Cellular & SPA

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TOM TOM pipeline pipeline 3D 3D Picking 3D alignment Averaging

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central slice + + + + + + + +

Tomographic reconstruction Tomographic reconstruction of 26S

• f 26S proteasomes

proteasomes

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• 0.1

0.2 0.4 0.6 0.8 1 nm

• 1

FSC coefficients c

• 180
• 120
• 60

60 120 180 90 30 60 120 150 180 Theta [°] Psi [°] a

Ice layer

Angular distribution of 26S Resolution FRCC

Nickell et al., BBRC (2007)

Non Non-

• Random Distribution of 26S in ice

Random Distribution of 26S in ice

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Averaging Averaging of tomographic

• f tomographic reconstructions

reconstructions

• f
• f the

the 26S proteasome 26S proteasome

first template (cylinder) sub tomograms (in total 153) average final average iterative alignment

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Nickell et al., BBRC (2007)

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Nickell et al., FEBS Letters (2007)

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Grid Grid scan scan: zoom : zoom

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High High-

• throughput

throughput data data acquisition acquisition

~100 images/hour total 800 images

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Particle Particle Picking Picking

tom_av2_particlepickergui

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2D 2D alignment alignment

tom_av2_alignment

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Overview Overview and and class class averages averages

Nickell et al., FEBS Letters (2007)

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Nickell et al., BBRC & FEBS Letters (2007)

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Multiclass Multiclass Analysis Analysis

tom_pcagui

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Eigenimage Gallery Eigenimage Gallery

tom_pcagui

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143 particles 114 particles

Two class averages of Ub Two class averages of Ub-

• Gold labelled 26S

Gold labelled 26S

difference

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A Titan A Titan… …

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Acknowledgments Acknowledgments

EM Jürgen Plitzko Günter Pfeifer

and Wolfgang Baumeister

26S Proteasome Stephan Nickell Florian Beck Oana Mihalache Inga Wolf Andreas Korinek Reiner Hegerl Ohad Medalia