Crystallographic mapping

An experiment with an irreversible inhibitor should carry with it a control experiment involving the addition of a substrate if the location of the reaction with inhibitor is at the active site, then the addition of a substrate will slow down the rate of inhibition. For example, the reactivity of papain (5 pM) with a 1.71 pM solution of 4-toluenesulphonylamidomethyl chloromethyl ketone suffers a drop of 1.68-fold when the substrate (methyl hippurate) is changed from 12.7 to 21.1 mM. The inhibitor which reacts covalently with the enzyme should carry either a radioactive or spectroscopic tag which would enable the location of the altered amino acid to be determined in the sequence, and hence in the three-dimensional X-ray crystallographic map of the enzyme. An alternative approach is to design an inhibitor with groups (analogous to those attached to the substrate) which force it to bind at the active site (Scheme 11.18). 

The center-to-center distance from either of the BChls of P to the nearest heme in the cytochrome subunit is about 21 A. A tyrosine residue of the protein sits squarely in the path from the heme to P [102]. Because the complete amino acid sequence of the cytochrome subunit has not yet been fitted to the crystallographic map of the reaction center, it is not clear which two of the four hemes are the low-potential hemes, and which two the high-potential, but information on this point should be available shortly. 

Hays FA, Teegarden AT, Jones ZJR, Harms M, Raup D, Watson J, Cavaliere E, Ho PS. How does sequence define structure A crystallographic map of DNA structure and conformation. Proc. Natl. Acad. Sci. U.S.A. 2005 102 7157-7162. 

The comparison with experiment can be made at several levels. The first, and most common, is in the comparison of derived quantities that are not directly measurable, for example, a set of average crystal coordinates or a diffusion constant. A comparison at this level is convenient in that the quantities involved describe directly the structure and dynamics of the system. However, the obtainment of these quantities, from experiment and/or simulation, may require approximation and model-dependent data analysis. For example, to obtain experimentally a set of average crystallographic coordinates, a physical model to interpret an electron density map must be imposed. To avoid these problems the comparison can be made at the level of the measured quantities themselves, such as diffraction intensities or dynamic structure factors. A comparison at this level still involves some approximation. For example, background corrections have to made in the experimental data reduction. However, fewer approximations are necessary for the structure and dynamics of the sample itself, and comparison with experiment is normally more direct. This approach requires a little more work on the part of the computer simulation team, because methods for calculating experimental intensities from simulation configurations must be developed. The comparisons made here are of experimentally measurable quantities. 

Oldfield TJ. Pattern-recognition methods to identify secondary structure within X-ray crystallographic electron-density maps. Acta Cryst. 2002 058 487-93. 

The structure was refined by block-diagonal least squares in which carbon and oxygen atoms were modeled with isotropic and then anisotropic thermal parameters. Although many of the hydrogen atom positions were available from difference electron density maps, they were all placed in ideal locations. Final refinement with all hydrogen atoms fixed converged at crystallographic residuals of R=0.061 and R =0.075. 

Because of the limitation intrinsic to the adoption of an explicit parametrised density model, many crystallographers have been dreaming of disposing of such models altogether. The thermally-smeared charge density in the crystal can of course be obtained without an explicit density model, by Fourier summation of the (phased) structure factor amplitudes, but the resulting map is affected by the experimental noise, and by all series-termination artefacts that are intrinsic to Fourier synthesis of an incomplete, finite-resolution set of coefficients. 

A better idea of the real size of an ion in a molecule can now be obtained from a study of electron density distributions, which it has recently become possible to obtain from accurate X-ray crystallographic studies of crystals. Figure 2.2 shows a contour map of the electron density distribution obtained in an X-ray crystallographic study of crystalline sodium chloride. The position of minimum electron density between two adjacent ions seems to be... 

The result is the electron density map of the protein crystal. The final task for the crystallographer is to build the appropriate protein model, i. e., putting amino acid for amino acid into the electron density. Routinely the theoretical amplitudes and phases are calculated from the model and compared to the experimental data in order to check the correctness of model building. The positions of the protein backbone and the amino acid side chains are well defined by X-ray structures at a... 

Fig. 6 Conformational map for benzophenones.

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