Section 19.1.5. Evaluation of correctness

It is an important first step in the structural analysis to determine the quality of the phases derived from the X-ray structure (Kossiakoff, 1983). Several methods have been used. Using the initial phasing model, the most powerful tests examine an unbiased neutron Fourier map for the appearance of features that are independent of the model. The presence or absence of these features, especially those resulting from the scattering of hydrogen and deuterium atoms, is the most reliable measure of the phasing model. One such test is to evaluate the appearance of the water structure, i.e., the water molecules hydrogen-bonded to the surface of the protein. The water molecules observed in the X-ray analysis are excluded from the neutron-phasing model. The test is applied in cases where the crystals have been soaked in D₂O. The peaks in the neutron density map that correspond to the strongly coordinated water-molecule positions owe their existence solely to the neutron data and phasing model. Even at an early stage, because of the large neutron-scattering potential of D₂O, many of these tightly bound waters found in the X-ray structures should also be observable in the neutron density map.

Another aspect to test phasing reliability is the ability to identify the orientation of side-chain amide groups of asparagine and glutamine. The difference in neutron scattering between O and the two deuteriums and the Nδ2 (5.8 f versus 22.6 f) is large enough to be detectable in the Fourier map when these groups are well ordered (Fig. 19.1.5.1). The use of unexchangeable hydrogens for evaluation is considerably more complicated, despite the fact that they constitute about one-half the total number of atoms in the molecule. The difficulty arises from the negative scattering character of the hydrogens, which displaces their apparent positions in the Fourier map from the true positions and, coupled with their short bond lengths, complicates the interpretation of the results. Additionally, it has been shown that small errors in positional and thermal parameters of the parent atoms can further complicate the identification of hydrogen-atom positions (Kossiakoff & Spencer, 1981).

Figure 19.1.5.1| top | pdf | Difference map of Asn34 in the trypsin structure. (a) In a protein X-ray analysis, the difference in scattering intensity between O and NH2 is much too small to be detected. In contrast, the neutron-scattering magnitudes of oxygen and nitrogen (5.8 f versus 9.4 f) are quite dissimilar, and there is additional scattering at the nitrogen site from the two bound deuterium atoms. The resulting differential is over 350%, quite large enough to be detected for well ordered side chains. The nitrogen and oxygen positions shown are from the X-ray model. The difference density indicates that the orientation of the nitrogen and oxygen atoms is incorrect and should be rotated by 180° around the Cβ–Cγ bond. (b) Difference map for Ser139. On well ordered hydroxyl side chains, the orientation of deuterium atoms can sometimes be assigned.