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

Max Planck Institute of Biochemistry Martinsried, Germany MAX PLANCK SOCIETY TOM TOM A toolbox toolbox for for Cryo Cryo- -Electron Electron A Tomography and Single Particle Tomography and Single Particle Analysis Analysis Stefan Bohn Stefan Bohn 15th of November 2007

Aquisition Alignment Reconstruction Aquisition Alignment Reconstruction Analysis Analysis ...more more than than 200 200 ... programs programs ...more than 150 ...more than 150 labs use it labs use it worldwide worldwide www.biochem.mpg.de/tom www.biochem.mpg.de/tom MPI of Biochemistry

Protein breakdown breakdown by by the the ubiquitin ubiquitin- -proteasome proteasome pathway pathway Protein MPI of Biochemistry

Co- -star: the 26S and 20S proteasome star: the 26S and 20S proteasome Co 26S Proteasome 19S cap 19S cap • A multi-subunit (>32) complex 20S core 20S core 19S cap 19S cap • 2.5 MDa • Degrades ubiquitinated substrates in an ATP-dependent manner 20S Proteasome β β β α β α 11nm α α • The proteolytic core complex • Conserved architecture from archaea to humans 15nm MPI of Biochemistry

The 20S proteasome of T. acidophilum T. acidophilum The 20S proteasome of 10 nm 10 nm side view view top view view side top Loewe et. al.,Science 268 Loewe et. al.,Science 268 (1995) (1995) MPI of Biochemistry

Gold labelled labelled Ubiquitin Ubiquitin- -aldehyde aldehyde binds binds 26S 26S specifically specifically Gold Hölzl et al., 2000 MPI of Biochemistry

MPI of Biochemistry

Integration of tomography and SPA in TOM Integration of tomography and SPA in TOM Tomography Single Particle Analysis Cellular & SPA Semi-Automated Acquisition Projection Alignment Reconstruction Volume Visualization Particle picking Particle Alignment Classification 3D reconstruction 3D reconstruction MPI of Biochemistry

Picking TOM pipeline pipeline 3D 3D TOM 3D alignment Averaging MPI of Biochemistry

Tomographic reconstruction of 26S of 26S proteasomes proteasomes Tomographic reconstruction + + + + + + + + central slice MPI of Biochemistry

Non- -Random Distribution of 26S in ice Random Distribution of 26S in ice Non Psi [°] - 180 - 120 -60 0 60 120 180 180 1 c 150 0.8 120 0.6 Theta [°] 90 FSC coefficients 60 0.4 30 0.2 0 0 Ice layer -0.1 0 7.8 3.9 2.6 1.95 a - 1 nm Angular distribution of 26S Resolution FRCC Nickell et al., BBRC (2007) MPI of Biochemistry

Averaging of tomographic of tomographic reconstructions reconstructions Averaging of the the 26S proteasome 26S proteasome of first template sub tomograms average final average (cylinder) (in total 153) iterative alignment MPI of Biochemistry

BBRC (2007) Nickell et al., MPI of Biochemistry

MPI of Biochemistry

Nickell et al., FEBS Letters (2007) MPI of Biochemistry

scan: zoom : zoom MPI of Biochemistry Grid scan Grid

High- -throughput throughput data data acquisition acquisition High ~100 images/hour total 800 images MPI of Biochemistry

tom_av2_particlepickergui Particle Picking Picking Particle MPI of Biochemistry

2D alignment alignment tom_av2_alignment 2D MPI of Biochemistry

Nickell et al., FEBS Letters (2007) class averages averages and class Overview and Overview MPI of Biochemistry

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

MPI of Biochemistry

Multiclass Analysis Analysis tom_pcagui Multiclass MPI of Biochemistry

Eigenimage Gallery Eigenimage Gallery tom_pcagui MPI of Biochemistry

Two class averages of Ub- -Gold labelled 26S Gold labelled 26S Two class averages of Ub 143 particles 114 particles difference MPI of Biochemistry

A Titan… … A Titan MPI of Biochemistry

Acknowledgments Acknowledgments 26S Proteasome EM Stephan Nickell Jürgen Plitzko Florian Beck Günter Pfeifer Oana Mihalache Inga Wolf Andreas Korinek Reiner Hegerl Ohad Medalia and Wolfgang Baumeister MPI of Biochemistry