From Electron Microscopy Maps to Atomic Structures Using Normal Mode-Based Fitting
Electron microscopy (EM) has made possible to solve the structure of many proteins. However, the resolution of some of the EM maps is too low for interpretation at the atomic level, which is particularly important to describe function. We describe methods
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. Introduction In the course of the last decade, important advances in the field of cryo-electron microscopy (EM) have lead to the solution of many low-resolution structures of proteins and biomolecular complexes (1). Since the elucidation of the function of these molecules and complexes often requires structural information at the atomic level, various modelling techniques have been developed that create atomic-resolution models compatible with the low-resolution structures by combining the cryo-EM data with atomic-level information from other sources. The first techniques of this kind took atomic-level structures for subdomains of a protein or for Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_13, © Springer Science+Business Media, LLC 2010
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individual molecules of a complex and placed them into the low-resolution structure of the assembly by applying rigid-body motions (2–8). However, the available atomic structures often correspond to different conformations than the cryo-EM structures. This observation leads to the development of flexible docking and fitting techniques. Most of these methods use the normal modes of an elastic network model to describe the flexibility of the molecules (9–12), but simulation techniques based on geometric constraints have also been used (13). In this chapter, we present a series of methods that make it possible to obtain an atomic structure from an EM map, given an atomic structure for a different conformation of the same protein. We show not only how to obtain an atomic structure, but also how to analyse the conformational change from the initial to the fitted atomic structure, and how to assess the quality of the fitted model. We demonstrate these methods using the sarco/endoplasmic reticulum Ca-ATPase (SERCA) as an example. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a transmembrane protein that belongs to a large family of P-type ATPases (14). This 100-kDa protein uses ATP as energy source to transport calcium and forms a stable aspartyl-phosphoryl intermediate that distinguishes it from F- and V-type ATPases, which are ATP synthases. The catalytic cycle consists of several intermediates that alternate steps of ion and nucleotide binding/release (Fig. 1). The calcium binding step required for enzyme activation permits the ATP terminal phosphate to be transferred to the enzyme. Then, calcium becomes occluded and vectorial transport occurs followed by hydrolytic cleavage of covalent phospho-intermediate. Proton countertransport from the lumen of reticulum is associated with calcium transport from the cytosol (15, 16) with a stoïchiometry of 1–1.5 leading to electrogenic transport (17). The structures of several intermediates have been defined by X-ray crystallography and EM (18, 19). Calcium binding sites reside within the membrane-inserted region formed by ten transmembrane helices. The ATP and catalytic sites are in the cytoplas
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