Phase refinement improvement procedure

Unfortunately, the relations between the electron density, the restraints we have discussed here, and the structure factors are non-linear. Thus, the only strategy we can adopt is to use the approximate phases we start out with and improve these iteratively. Even this is not straightforward, mainly because Eq. 1 is expensive to compute. However, there exists a powerful and straightforward procedure that is used in virtually all phase refinement programs Eourier cycling. 

Model building and refinement are both phase improvement procedures... 

The above discussions have shown how selected analytical techniques can be applied to vastly different proteins to solve a myriad of problems. These include routine assays amino acid and sequencing analyses specialized techniques FAB-MS and IEF conventional techniques refined to improve their utility reversed-phase HPLC using different pHs, organic modifiers, and temperatures and chemical and enzymatic modifications. The latter two procedures have been shown to be effective not only in elucidating primary structure but also in probing the conformation of proteins. 

An alternative procedure, called molecular replacement, uses information about known structures that are believed to be similar to that of the species being investigated. The known structure is used to estimate the electron density of the unknown structure, which is then refined and improved. Another method of dealing with the phase problem is to introduce atoms which absorb radiation in the region of the incident X-rays, leading to a process called anomalous scattering . For proteins, a popular method is to replace S by Se by using selenomethionine in place of methionine. For nucleic acids, iodouracil or iodocytosine can be used in place of thymine and cytosine respectively. 

The ease with which a molecular model can be fitted to the observed density depends on the resolution at which the map is calculated (compare figures 2.1(c), 2.1 (d), 10.4 and 10.5) and its quality. The resolution limit of the calculation is set by the phase determination method. For the method of isomorphous replacement, phasing is successful usually to =2.5-3.0 A. In the case where a related structure is already known the method of molecular replacement (Rossmann (1972)) can be used whereby rotation and translation matrices are determined and then calculated phases used. Clearly, these two procedures are both approximate methods. The model is usually improved by using least-squares methods of refinement (for a collection of papers see Machin, Campbell and Elder (1980)) and higher resolution data (better than 2 A or so). Refinement methods involve the determination of shifts to the atomic parameters (coordinates and thermal parameters) so as to agree better with the observed diffraction data whilst preserving the known stereochemical features of proteins and nucleic acids. This is achieved by minimising a composite observational function ... 

A recent analytical procedure by Flores et al. targeted the optimization of the interface performance in the online coupling of reversed phase liquid chromatography and gas chromatography (RPLC-GC) coupling by means of a horizontally positioned PTV (Programmed Temperature Vaporizer) injector. It improved the sensitivity achievable in the direct analysis of olive oil-mixtures with 5% or 12% of some virgin and refined hazelnut oils, respectively, based on the analysis of fil-bertone enantiomers within 30 min and without any kind of pretreatment. 

To secure a uniform film deposition from the solution of a stationary phase, the inner column s surface is first treated by an organic compound, the task of which is to improve the surface wettability and to mask potentially adsorptive column sites. A variety of stationary phases can now be successfully coated for capillary GC with a controlled film thickness. Refined procedures now exist even for the chemical immobilization of some stationary liquids. 

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