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Free keywords:
HUMIDITY CONTROL; IMPROVING DIFFRACTION; DEHYDRATION; CRYSTALLIZATION;
MECHANISM; KINASE; POWER
Abstract:
A method and the design of instrumentation, and its preliminary practical realisation, including test experiments, with the object of inducing phase changes of biomolecular crystals by controlled dehydration through heating with infrared (IR) light are described. The aim is to generate and select crystalline phases through transformation in the solid state which have improved order (higher resolution in X-ray diffraction experiments) and reduced mosaic spread (more uniformly aligned mosaic blocks) for diffraction data collection and analysis. The crystal is heated by pulsed and/or constant IR laser irradiation. Loss of crystal water following heating and its reabsorption through equilibration with the environment is measured optically by a video system. Heating proved superior to traditional controlled dehydration by humidity change for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein kinase). Heating with IR light is experimentally simple and offers an exploration of a much broader parameter space than the traditional method, as it allows the option of varying the rate of phase changes through modification of the IR pulse strength, width and repeat frequency. It impacts the crystal instantaneously, isotropically and homogeneously, and is therefore expected to cause less mechanical stress.
