2/11/09 Imaging gaps in biology and medicine Computing the - PDF document

2/11/09 Imaging gaps in biology and medicine Computing the molecular structures of cells and viruses using 3D electron microscopy Sriram Subramaniam David Goodsell TIBS (1991) Subramaniam, Curr. Opin. Microbiol. (2005) From molecules to cells Images of single multi-enzyme complexes ~50 nm ~2000 nm Dynamic multiprotein Signaling assemblies complexes in intact cells ~150 nm ~30,000 nm 500 Å HIV structure and cell Subcellular architecture of entry mechanisms melanoma cells Goodsell, TIBS (1991) Reconstructed 3D structure…. Reconstructed 3D structure…. Structure and function of pyruvate dehydrogenase 50 nm ….and atomic interpretation pyruvate + CoA acetyl CoA Milne et al 2002, 2004, 2006; Borgnia et al 2004; Lengyel et al 2008 1

2/11/09 From 2D to 3D Electron microscopes as tools for 3D imaging From cells to molecules: Bridging the gap 1000 nm 5 nm Projection images Tomogram 100,000 nm (from Macnab and colleagues) 50 nm 3D averaging of receptors to determine structure Direct visualization of receptor Direct 3D imaging of receptor clusters arrays in intact bacterial cells 100 nm Khursigara, Wu and Subramaniam J. Bacteriology (2008) Khursigara, Wu, Zhang, Lefman and Subramaniam Zhang, Khursigara, Hartnell and Subramaniam Proc. Natl. Acad. Sci. USA (2008) Proc. Natl. Acad. Sci. USA (2007) Structural biology of signaling in single cells! From structure to physiology Yet, the major goal of HIV /AIDS research eludes us: Expanded Compact the development of a safe and effective HIV vaccine, our best hope for ultimately ending the pandemic. The search for a vaccine has been made extremely dif fi cult by the nature of the virus, particularly its ability to integrate into the genome of host cells, to mutate Predicting behavior +methylation readily and to conceal that part of its outer coat that From 3D image would induce protective antibodies. +serine Khursigara, Wu, Zhang, Lefman and Subramaniam Dr. Anthony Fauci in Nature , May 2008 Quantitative modeling Proc. Natl. Acad. Sci. USA (2008) Network maps Receptor distribution plots 2

2/11/09 Structural analysis of viral spikes by electron tomography Cryo-electron microscopy of “symmetric” vs. “asymmetric viruses Each viral particle identical; can achieve resolutions better than ~4 Å by 3D averaging Each viral particle 2D image different; missing data distorts features depending on orientation tomogram From Subramaniam et al Curr. Opin. Struct. Biol. 3D volume (2007) HIV-1 surface spike in complex with neutralizing antibody HIV-1 surface spike in complex with CD4 and 17b gp41 V1/V2 Map at 20 Å resolution Map at 20 Å resolution Most objects of biological interest are Catching HIV in the act Destructive imaging: Ion abrasion SEM thicker than 500 nm… 10 1 10 4 10 8 10 12 10 16 10 20 10 24 10 28 Daltons We therefore need methods that extend beyond Liu, Bartesaghi, Borgnia, Sapiro and Subramaniam conventional transmission electron microscopy Nature (2008) “Slice and View” 3

2/11/09 Heymann, Hayles, Giannuzzi, Lich and Subramaniam J. Struct. Biol. (2006) Diatoms! Destructive imaging: Ion abrasion SEM *Third place fi nish, behind “Human bloodstream” and “Visualizing the bible” Bliss and Subramaniam MNT-1 melanoma cell (z-pixel 30 nm; x,y pixel 6 nm) “Melanoma cell in 3D” Science Sept. 25, 2008 issue Heymann, Shi, Kim, Bliss, Milne and Subramaniam J. Struct. Biol. (2009) in press Images from Round et al., 1990 Walking into dividing diatoms Thank you Biowulf! 4