What is X-ray Crystallography ? X-ray crystallography is an experimental technique that exploits the fact that X-rays are diffracted by crystals. It is not an imaging technique. X-rays have the proper wavelength (in th Å t ö (i the Ångström range, ~10-10 m) t be scattered by the electron cloud of an atom of 10 10 ) to b tt d b th l t l d f t f comparable size. Based on the diffraction pattern obtained from X-ray scattering off the periodic assembly of molecules or atoms in the crystal, the electron density can be reconstructed. Additional phase information must be extracted either from the diffraction data or from supplementing diffraction experiments to complete the reconstruction (the p phase problem in crystallography). A model is then progressively built into the p y g p y) p g y experimental electron density, refined against the data and the result is a quite accurate molecular structure. Why Crystallography ? The knowledge of accurate molecular structures is a prerequisite for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents and drugs. Crystallography can reliably provide the answer to many structure related questions, from global folds to atomic details of bonding. In contrast to NMR, which is an indirect spectroscopic method, no size limitation exists for the molecule or complex to be studied. The price for the high accuracy of crystallographic structures is that a good crystal must be found, and that only limited information about the molecule's dynamic behavior is available from one single diffraction experiment.
Outline of the experiment
In a macromolecular X-ray diffraction experiment a small protein crystal is placed into an intense X-ray beam and the diffracted X-rays are collected with an area detector (it is advantageous to cool the crystals to low temperatures, primarily to prevent radiation damage). damage) The diffraction pattern consists of reflections of different intensity, and a lot of intensity patterns need to be collected to cover all necessary crystal orientations. After some data processing, we end up with a list of indexed reflections and their intensities.
The diffracted X-rays are scattered by the crystal at a certain angle. The further backwards the x-rays scatter, the higher we say is the resolution of the data set. The extent to which the crystal diffracts determines how fine a detail we can actually distinguish in our final model of the structure. High resolution is thus desirable. Knowing the wavelength and the diffraction angle of a reflection, its resolution d can be easily calculated :
. This is just a reformulation of the famous Bragg equation
X-ray Diffraction Equipment
The Experimental Setup
To perform an X-ray diffraction experiment, we need an x-ray source. In most cases a rotating anode generator producing an X t ti d t d i X-ray b beam of a characteristic wavelength is used. f h t i ti l th i d Intense, tunable X-ray radiation produced by a Synchrotron provides additional advantages. The primary X-ray beam is monochromated by either crystal monochromators or focusing mirrors. After the beam passes through a helium flushed collimator it passes through the crystal mounted on a pin on a goniometer head. The head is mounted to a goniometer which allows to position the crystal in different orientations
in the beam. The diffracted X-rays are recorded using image plates, Multiwire detectors or CCD cameras.
Flash cooling protein crystals to cryogenic temperatures (~100 K) offers many advantages, the most significant of which is the elimination of radiation damage. A part of the X-rays passing through the crystal is scattered in different directions into a detector. The detector delivers an image of the diffraction spots. A large number of these images recorded from different crystal orientations are processed (scaled and merged) into a final list of indexed reflection intensities.
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