Two-wavelength MAD phasing and radiation damage: a case study.
Radiation damage affects MAD experiments in two ways: (i) increased absorption by the crystal at the wavelengths of interest for the experiment results in faster crystal deterioration; (ii) lack of isomorphism induced by radiation damage causes problems when scaling and merging data at different wavelengths and can prevent accurate measurement of anomalous and dispersive differences. In an attempt to overcome these problems in the case of radiation-sensitive crystals of vinculin, two-wavelength MAD data were collected at the Se absorption-edge inflection and at high-energy remote wavelengths. Although this strategy resulted in a lower total absorbed dose compared with a standard three-wavelength experiment using the peak wavelength, an increase in the unit-cell volume and other effects attributable to radiation damage were still observed. In an effort to extract the maximum information available from the data, different data-processing and scaling procedures were compared. Scaling approaches involving local scaling of unmerged reflections were consistently successful and most ordered Se sites could be located. Subsequent use of these sites for phasing resulted in an interpretable electron density map. This case demonstrates the feasibility of two-wavelength MAD in the presence of moderate radiation damage using conventional data collection strategies and widely available standard software.
González
Ana
A
Stanford Linear Accelerator Center, Menlo Park, CA 94025, USA.
von Delft
Frank
F
Liddington
Robert C
RC
Bakolitsa
Constantina
C
D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D005240:Feasibility Studies; D011487:Protein Conformation / Q000528:radiation effects; D012984:Software; D016596:Vinculin / Q000737:chemistry* / Q000528:radiation effects*
D016596:Vinculin