Structure determination, experimental microscopy

A completely new method of determining siufaces arises from the enormous developments in electron microscopy. In contrast to the above-mentioned methods where the surfaces were calculated, molecular surfaces can be determined experimentally through new technologies such as electron cryomicroscopy [188]. Here, the molecular surface is limited by the resolution of the experimental instruments. Current methods can reach resolutions down to about 10 A, which allows the visualization of protein structures and secondary structure elements [189]. The advantage of this method is that it can be apphed to derive molecular structures of maaomolecules in the native state. 

Current understanding of adsorption on Si(lOO) is summarized below. More detailed reviews are readily available. The structure of the clean surface has been determined experimentally by electron diffraction [9] and x-ray diffraction [10]. Liu and Hoffman [11] have given a concise review of the geometry and electronic structure of the bare surface. The classic review of Appelbaum and Hamann [12] describes the electronic structure of the bare surface and the bonding of H atoms. Accurate diffraction studies of the H-covered surfaces are not available. Infrared spectroscopy of H-covered Si surfaces has been reviewed by Chabal et al. [13], and the tunneling microscopy of these systems has been reviewed by Boland [14]. 

Although experimental studies of DNA and RNA structure have revealed the significant structural diversity of oligonucleotides, there are limitations to these approaches. X-ray crystallographic structures are limited to relatively small DNA duplexes, and the crystal lattice can impact the three-dimensional conformation [4]. NMR-based structural studies allow for the determination of structures in solution however, the limited amount of nuclear overhauser effect (NOE) data between nonadjacent stacked basepairs makes the determination of the overall structure of DNA difficult [5]. In addition, nanotechnology-based experiments, such as the use of optical tweezers and atomic force microscopy [6], have revealed that the forces required to distort DNA are relatively small, consistent with the structural heterogeneity observed in both DNA and RNA. 

There are three potential methods by which a protein s three-dimensional structure can be visualized X-ray diffraction, NMR and electron microscopy. The latter method reveals structural information at low resolution, giving little or no atomic detail. It is used mainly to obtain the gross three-dimensional shape of very large (multi-polypeptide) proteins, or of protein aggregates such as the outer viral caspid. X-ray diffraction and NMR are the techniques most widely used to obtain high-resolution protein structural information, and details of both the principles and practice of these techniques may be sourced from selected references provided at the end of this chapter. The experimentally determined three-dimensional structures of some polypeptides are presented in Figure 2.8. 

First of all, the behavior of the enzymes in the membrane differs markedly from the behavior of the unbound enzymes in solution. It is pertinent to note that the medium in which the enzyme bound to a membrane acts might be determined not only by the composition and structure of the membrane itself, but also by the local concentration distribution of substrate and products. The microenvironment in the membranes is the result of a balance between the flow of matter and enzyme reactions. The substrate and product concentrations in the membrane differ from point to point across the membrane and also from those at the outer solution. By electron microscopy this was experimentally demonstrated beyond doubt with the DAB-peroxidase system by Barbotin and Thomas.16 The effects of these profiles were studied with... 

As reported previously, the morphology of fibrous cellulose-polyvinyl copolymers, determined by electron microscopy, depends on the method of free-radical initiation of the copolymerization reaction, the experimental conditions during the reaction, and the type of vinyl monomer used. Variations in the shape of the fibrous copolymer cross section, in layering effects in the copolymer structure, and in location and distribution of the polyvinyl polymer within the fibrous structure were shown (1,2,7,29,52). 

How can we experimentally determine which is the outer and which the inner phase One possibility is to use electron microscopy which provides detailed images of the emulsion structure. Electron microscopes are relatively expensive and sample preparation requires time and skill. Therefore alternative techniques are often used ... 

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