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.