Structure Determination of Membrane Protein by Both Cryo-Electron Tomography and Single Particle Analysis
The structure determination of membrane protein in lipid environment can be carried out using cryo-electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of me
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. Introduction Electron microscopy (EM) and cryo-electron microscopy (cryo-EM) are used to image ultrastructure of tissues and cells as well as to provide information on the structure of purified molecules including membrane proteins. Cryo-EM enables the visualization of the specimen embedded in amorphous ice that represents a state close to the native state. To study membrane protein in a lipid environment, structural analysis is performed on membrane protein assembled in twodimensional (2D) arrays either occurring naturally or induced after reconstitution into a lipid membrane. Unilamellar 2D arrays of membrane protein are amenable to electron crystallography studies. Instead, stacks of 2D arrays protein or multilamellar crystal are less suitable mainly because of complications to characterize 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_11, © Springer Science+Business Media, LLC 2010
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Trépout, Taveau, and Lambert
the unit cell and process the data, the number of layers and their geometry being difficult to estimate despite few attempts (1, 2). Both single particle analysis and cryo-electron tomography (cryo-ET) are also useful approaches for the structural analysis of biological components by transmission EM. Classically, single particle analysis enables to determine the three-dimensional (3D) structures of relatively large proteins and macromolecular complexes from a large set of images, assuming that all the samples have the same architecture. Single particle analysis needs to determine the Euler angles of each particle randomly oriented in ice. Then the final 3D density map is computed not from a single particle but from thousands of particles. With this method, the specimen is illuminated only once, but the determination of the Euler angles is a key issue (random conical tilt method, common line approach, etc.). Purified membrane proteins or assemblies into small regular structures have been studied by 3D negative-stain EM and cryo-EM using single particle method (3 see references inside; 4 –7). There is no notable difference between membrane protein and soluble protein methodologies except the fact that the detergent associated to the membrane protein and the detergent in solution also contribute to the images that require careful attention for their interpretation especially in the membrane region. Electron tomography (ET) is often used to visualize 3D subcellular structures and supramolecular assemblies (8) at molecular resolution after collecting a tilt series of a unique object. In ET, the specimen is illuminated multiple times from various different view angles to compute a 3D structure. Examples of recent applications of ET to negatively stained supramolecular assemblies include studies of fibrillin-rich microfibrils and the corneal collagen fibril structure (9, 10) or frozen-hydrated proteoliposome– bacteriophage complexes (11) leading to the overall description of the asse
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