Electron Microscopy and Image Processing: An Essential Tool for Structural Analysis of Macromolecules

Abstract

Macromolecular electron microscopy (EM) deals with macromolecular complexes and their placement within the cell-linking the molecular and cellular worlds as a bridge between atomic-resolution X-ray crystallographic or NMR studies and lower resolution light microscopy. The amount of specimen required is typically 10(2) to 10(3) times less than for X-ray crystallography or NMR. Electron micrographs of frozen-hydrated specimens portray native structures. Computer averaging yields enhanced images with reduced noise. Three-dimensional reconstructions may be computed from multiple views. Under favorable circumstances, resolutions of 7 to 10 A are achieved. Fitting atomic-resolution coordinates of components into three-dimensional density maps gives pseudo-atomic models of a complex's structure and interactions. Time-resolved experiments describe conformational changes. Electron tomography allows reconstruction of pleiomorphic complexes and sub-cellular structures. Electron crystallography has produced near-atomic resolution models of two-dimensional arrays, notably of membrane proteins.

In-vivoandin-vitrotechniques used to investigate Alzheimer's disease

“…Negative staining, rotary shadowing and cryo-electron microscopy have been used to exchange the image and contrast. Steven and Belnap (2005) used TEM to characterize various assembly states of Aβ, including low molecular weight Aβ (Bitan et al 2003a(Bitan et al , 2003b, small Aβ oligomers (Roher et al 1996;Modler et al 2003), paranuclei (Bitan et al 2003a(Bitan et al , 2003b, protofibrils (Roher et al 1996;Walsh et al 1999;Ward et al 2000;Nichols et al 2002;Kheterpal et al 2003) and fibrils (Wood et al 1996;Ward et al 2000). Cryo-electron microscopy has been used to resolve the structure of Aβ42 fibrils, which comprise regularly spaced ( ∼ 45 Å) protofilaments that twist regularly along the fibril axis (Luhrs et al 2005).…”

Life Inside a Thin Section: Tomography

“…Resolution and contrast of details are necessarily poor compared to the images derived from structures stained with heavy metals, which produce much more electron scattering and a higher signal to noise ratio. However, cryo-EM tomography has enjoyed important successes in studying the high resolution structure of individual proteins or isolated protein complexes where large number of structures can be averaged (Frank, 1981; Steven and Belnap, 2005; Zhang et al, 2008). The difficulties with cryo-EM arise in directly studying neuronal structures, such as synapses, which are intrinsically heterogeneous.…”