| X-ray Crystallography |
Article Index for X-ray |
Shopping X-ray |
Website Links For Crystallography |
Information AboutX-ray Crystallography |
| CATEGORIES ABOUT X-RAY CRYSTALLOGRAPHY | |
| crystallography | |
| diffraction | |
| x-rays | |
| protein structure | |
| protein methods | |
| synchrotron related techniques | |
|
INORGANIC & SIMPLE ORGANIC STRUCTURES In inorganic chemistry, x-ray crystallography is used to determine lattice structures as well as chemical formulas, bond lengths and angles. The primary methods used in inorganic structures are powder diffraction and single-crystal diffraction. Single Crystal Diffraction Many complicated inorganic and organometallic systems have been analyzed using single crystal methods, such as fullerenes, metalloporphyrins, and many other complicated compounds. Single crystal is also used in pharmaceutical industry, due to recent problems with Polymorphs . The major limitation to the quality of single-crystal data is crystal quality. Inorganic single-crystal x-ray crystallography is commonly known as small molecule crystallography, as opposed to macromolecular crystallography. Powder Diffraction X-ray powder diffraction finds frequent use in Materials Science because sample preparation is relatively easy, and the test itself is often rapid and non-destructive. The vast majority of engineering materials are crystalline, and even those which are not yield some useful information in diffraction experiments. The pattern of powder diffraction peaks can be used to quickly identify materials (thanks to the JCPDS pattern database), and changes in peak width or position can be used to determine crystal size, purity, and Texture . Biological structures The first Protein crystal structure was of Sperm Whale Myoglobin , as determined by Max Perutz and Sir John Cowdery Kendrew in 1958, which led to a Nobel Prize In Chemistry . The X-ray diffraction analysis of myoglobin was originally motivated by the observation of myoglobin crystals in dried pools of blood on the decks of whaling ships. Today X-ray crystallography is used by Pharmaceutical Companies to determine specifically how Drug Lead Compounds interact with their protein targets. Biological X-ray crystallography is to date the most prolific discipline within the area of Structural Biology ; out of the ~35000 protein structures solved, X-ray crystallography is responsible for ~29000. Nuclear Magnetic Resonance has contributed almost 5000 and Electron Microscopy just over 100. Other Biophysical methods, such as IR Spectroscopy and Powder Diffraction make up the remaining structures, according to the Protein Data Bank (PDB). CRYSTALLISATION In order to solve a crystal structure, you must first Crystallise the compound of interest. This is because a single molecule in solution has insufficient scattering power alone. A crystal can be considered to be an (effectively) infinite repeating array of our molecule of interest. The Laue conditions and Bragg's Law show that constructive Interference between diffracted X-rays that are In-phase reinforce each other, so that the Diffraction pattern becomes detectable. The geometric conditions where diffraction occurs can be visualised using Ewald's Sphere . Crystallization of small molecules has traditionally followed three methods
Even though small molecules are relatively more facile to crystallize than macromolecules, there are many compounds reported that have failed to give diffraction quality crystals. Crystallisation of macromolecules is not trivial. Traditional methods of crystallising inorganic molecules have been modified to be gentle enough for proteins, which are sensitive to temperature and high concentrations of organic solvents.Many methods exist to crystallise proteins, but the two most successful methods are the ''microbatch'' and ''vapour diffusion'' techniques. Concentrated solutions of the protein are mixed with various solutions, which typically consist of:
In either ''microbatch'' or ''vapour diffusion'' the solutions are allowed to concentrate over time. In solutions of a favourable composition, the protein becomes supersaturated and ''crystal nuclei'' form, leading to crystal growth. Typically protein crystallographers can screen hundreds or thousands of conditions before a suitable condition is found that leads to a crystal of suitable quality. As a rule of thumb, some useful detail can be gained from a crystal that diffracts with a Resolution of better than 4 Angstrom s (400 Picometer s). Many biomolecules of interest still have not been successfully crystallised. Imperfections in the crystal structure, caused by impurities or sample contamination can prevent the acquisition of atomic Resolution images. Convection caused by temperature variations within the forming crystal can also cause imperfections, and one of the proposed scientific applications of the International Space Station is the growth of crystals, because convection is reduced in the free fall environment of an orbiting spacecraft. X-ray Diffraction Experiment Once prepared the crystals are harvested and often cryocooled with gaseous or liquid Nitrogen at a temperature of around 100 Kelvin s or −172 °C. Liquid Helium is occasionally used too, but it is often not necessary to cool crystals that much (and it also costs more). Cryocooling crystals both reduces radiation damage incurred during data collection and decreases thermal motion within the crystal, giving rise to better Diffraction limits and higher quality data. Crystals are then mounted on a Diffractometer coupled with a machine that emits a beam of X-rays This can either be a Rotating-anode Type Source or a Synchrotron . The X-rays are diffracted by their interaction with the Electrons in the crystal, and the pattern of diffraction is recorded on film or more recently Charge-coupled Device detectors and scanned into a computer. Successive images are recorded as a crystal is rotated within the X-ray beam. Data processing The data collected from a diffraction experiment is a Reciprocal Space representation of the crystal lattice. The position of each diffraction 'spot' is governed by the size and shape of the Unit Cell , and the inherent Symmetry within the crystal. The intensity of each diffraction 'spot' is recorded, and is proportional to the square of the ''structure factor'' Amplitude . The ''structure factor'' is a Complex Number containing information relating to both the Amplitude and Phase of a Wave . In order to obtain an interpretable ''electron density map'', we must first obtain phase estimates (An electron density map allows a crystallographer to build a starting model of our molecule) This is known as the Phase Problem can be accomplished in a variety of ways.
Having obtained initial phases we can build an initial model (our Hypothesis ) and then refine the Cartesian Coordinates of atoms and their respective B-factors (relating to the thermal motion of the atom) to best fit the observed diffraction data. This generates a new (and hopefully more accurate) set of phases and a new electron density map is generated. The model is then revised and updated by the crystallographer and a further round of refinement is carried out. This continues until the correlation between the diffraction data and the model is maximised. Once the model of a molecule's structure has been finalised, it is often deposited in a crystallographic database such as the Protein Databank or the Cambridge Structure Database . Many structures obtained in private commercial ventures to crystallise medicinally relevant proteins, are not deposited in public crystallographic databases. SEE ALSO
REFERENCES
|
|
|