The determination of label positions in membrane proteins by neutron and anomalous X-ray diffraction of powder samples

The applications of isomorphous labels for neutron and anomalous X-ray diffraction on powder samples in combination with phase and intensity information from electron microscopy (EM) are currently investigated. In particular, several groups have performed neutron diffraction on bacteriorhodopsin from Halobacterium halobium selectively deuterated at certain amino acids or at the chromophore retinal in order to show structural details which were not detectable from the EM data alone. In these investigations the difference Fourier method has been used to determine label positions. The limitation of this method with respect to the strength of the label is well known in X-ray single-crystal studies. In the present paper, it is shown to what extent the applicability of the method is weakened by the transfer of EM information to the neutron diffraction data when powder samples are used. The analysis is based on computer simulation experiments. A procedure is described to test the validity of the approach. With respect to neutron diffraction the method is applicable to data of moderate resolution when the relative steps in the density maps for neutron and electron diffraction are similar. Label positions can be obtained with an accuracy of ±1 Å from data of moderate resolution. In addition, it is shown how anomalous X-ray diffraction can be used in determining label positions using EM phases and intensity information. A procedure is described to obtain the correct label position, despite the fact that in this case the non-equal intensities I(+h) and I(-h) are superimposed on the same powder ring.