Structural information difference maps

Abstract To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues. 

As a result of the recognized role of transition metal hydrides as l reactive intermediates or catalysts in a broad spectrum of chemical reactions such as hydroformylation, olefin isomerization, and hydrogenation, transition metal hydride chemistry has developed rapidly in the past decade (J). Despite the increased interest in this area, detailed structural information about the nature of hydrogen bonding to transition metals has been rather limited. This paucity of information primarily arises since, until recently, x-ray diffraction has been used mainly to determine hydrogen positions either indirectly from stereochemical considerations of the ligand disposition about the metals or directly from weak peaks of electron density in difference Fourier maps. The inherent limi-... 

In the fifth step of an X-ray structure determination the electron density map is calculated using the intensities and phase information. This map can be thought of as a true three-dimensional image of the molecule revealed by the X-ray microscope. It is usually displayed as a stereoscopic view on a computer graphics system (Fig. 3-22). It is also often prepared in the form of a series of transparencies mounted on plastic sheets. Each sheet represents a layer, perhaps 0.1 ran thick, with contour lines representing different levels of electron density. 

Peaks occur in a difference map in positions in the unit cell where the model did not include enough electron density valleys appear in places where the model contained too much electron density. This information may be used to obtain more precise atomic positions, atomic displacement parameters, or atomic numbers. For example, in the last category, the identities of atoms (carbon or nitrogen) in a tricyclic molecule were established by setting all atoms to one type (carbon in this case) in the structure factor calculation. A difference map was calculated with the calculated phases and examined for excess electron density at atomic positions (Table 9.2). It was found to be possible to distinguish between nitrogen (seven electrons) and carbon (six electrons), even though these atoms are adjacent in the Periodic Table. 

Simulated annealing refinement is usually unable to correct very large errors in the atomic model or to correct for missing parts of the structure. The atomic model needs to be corrected by inspection of a difference Fourier map. In order to improve the quality and resolution of the difference map, the observed phases are often replaced or combined with calculated phases, as soon as an initial atomic model has been built. These combined electron density maps are then used to improve and to refine the atomic model. The inclusion of calculated phase information brings with it the danger of biasing the refinement process towards the current atomic model. This model bias can obscure the detection of errors in atomic models if sufficient experimental phase information is unavailable. In fact during the past decade several cases of incorrect or partly incorrect atomic models have been reported where model bias may have played a role [67]. 

Morals (1) Examine difference maps carefully in the late stages of refinement. (2) Do not accept spectral information if the resultant formulation leads to a structure that contains unprecedented features. 

Chemical nucleases provide detailed information concerning the interactions of regulatory proteins with DNA (12) and have recently been applied to intact transcription complexes. High-resolution probes such as hydroxyl radical-FeEDTA (192), as well as dimethyl sulfate (DMS) and DNAsel, were used to deconvolute the interactions of MerR, Hg-MerR, and RNA polymerase with Pr, the DNA sequences in the regulatory region (the promoter) of the mercuric ion responsive genes (145). These studies provide a structural map, also known as a footprint , of the interactions of both proteins with the DNA in complexes isolated at different stages in the activation process and provide structural information on the physical role Hg-MerR in the activation mechanism. Several of the kinetic inter-... 

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