Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 98, Issue 19, Pages 10648-10653Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.191003998
Keywords
MAD; protein crystallography; resonance scattering; x-ray absorption
Categories
Funding
- NIGMS NIH HHS [R01 GM034102, R37 GM034102, GM34102] Funding Source: Medline
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The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its M-IV (3.326 Angstrom) and M-V (3.490 Angstrom) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f ' = -70e, f = 80e at the M-IV edge and f ' = -90e, f = 105e at the Mv edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the M-IV edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-Angstrom Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of (0)(phiT) - (0)(phiA) for replicated determinations is 32 degrees) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.
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