Ambiguous electron density maps

There is also a conformational ambiguity characteristic of the histidine side chain as it is built into electron density maps The two possible orientations about the Cfi-Cy bond are indistinguishable, so the crystal-lographer must select one of these orientations based on chemical intuition [e.g., the availability of hydrogen bond interaction(s) in one particular orientation and not the other]. Additionally, the proper histidine tautomer is likewise selected by chemical intuition. Reynolds et al. (1973) demonstrated that the Ne-H tautomer is the predominant form of free histidine in solution, and these investigators showed that the ratio Ne-H tautomer/N8-H tautomer is approximately 80% 20% (Fig. 13). 

The degree of accuracy that is attained depends on both the quality of the data and the resolution. At low resolution, 4 to 6 A (0.4 to 0.6 nm), the electron density map reveals little more in most cases than the overall shape of the molecule. At 3.5 A, it is often possible to follow the course of the polypeptide backbone, but there may be ambiguities. At 3.0 A, it is possible in favorable cases to begin to resolve the amino acid side chains and, with some uncertainty, to fit the sequence to the electron density. At 2.5 A, the positions of atoms often can be fitted with accuracy of 0.4 A. To locate atoms to 0.2 A, a resolution of about 1.9 A and very well ordered crystals are necessary. 

The electron density distribution for solvent molecules can be improved if the contribution from bulk water to the X-ray scattering is included in the model. This affects the low-angle j X-ray intensity data which are omitted in early stages of the least-squares refinement of protein crystal structures. If they are included in refinement and properly accounted for, the signal-to-noise ratio in the electron density maps is significantly improved and the interpretation of solvent sites is less ambiguous. 

For the determination of the electron density map of a molecule the amplitudes and phases of the waves scattered by a single crystal are required for a number of Bragg reflections. Common X-ray techniques yield the product (Ah vh) (Ah 1waves scattered by the electrons of the molecule into the Bragg reflection H. Thus, the scattering amplitude Ah is obtained, but the phase information is lost. The solution of this phase problem for protein structure determination is based on Perutz and Kendrew s isomorphous replacement method (108-111). In this procedure Bragg reflections have to be measured at least three times, first on a crystal of native molecules, and then on two crystals, in which reference scatterers (for example Hg atoms) have been substituted at well-defined positions. From the difference of the measured intensities one can calculate the relative phases without ambiguity. 

To resolve the ambiguities in interpretation of the single-stranded regions in the electron density map, we used a variation of a technique which is very common in small molecule crystallography. Phases are calculated from model coordinates of a fragment of the structure which has been seen by the heavy atom method or the direct... 

After the calculation of MIR phases, the electron density was improved by solvent flattening followed by non-crystallographic symmetry averaging. The map after solvent flattening showed most of the secondary structure. However, the connectivity of electron density was ambiguous in the loop regions. Since it was clear that the asymmetric unit contained a trimer of GIF related by a non-crystallographic... 

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