期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 3, 页码 1094-1099出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0905481107
关键词
cryocrystallography; hydrogen abstraction; macromolecular crystallography; small-angle x-ray scattering; x-ray radiolysis
资金
- National Center of Excellence in Research (NCCR) Structural Biology program of the Swiss National Science Foundation
Radiation damage is the major impediment for obtaining structural information from biological samples by using ionizing radiation such as x-rays or electrons. The knowledge of underlying processes especially at cryogenic temperatures is still fragmentary, and a consistent mechanism has not been found yet. By using a combination of single-crystal x-ray diffraction, small-angle scattering, and qualitative and quantitative radiolysis experiments, we show that hydrogen gas, formed inside the sample during irradiation, rather than intramolecular bond cleavage between non-hydrogen atoms, is mainly responsible for the loss of high-resolution information and contrast in diffraction experiments and microscopy. The experiments that are presented in this paper cover a temperature range between 5 and 160 K and reveal that the commonly used temperature in x-ray crystallography of 100 K is not optimal in terms of minimizing radiation damage and thereby increasing the structural information obtainable in a single experiment. At 50 K, specific radiation damage to disulfide bridges is reduced by a factor of 4 compared to 100 K, and samples can tolerate a factor of 2.6 and 3.9 higher dose, as judged by the increase of R-free values of elastase and cubic insulin crystals, respectively.
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