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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
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 ~36000 protein structures solved, X-ray crystallography is responsible for ~30000. NMR spectroscopy 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 crystallize 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 protein solutions are allowed to concentrate over time. A common method for crystallisation is hanging drop vapour diffusion. A small droplet of concentrated protein- and precipitant-containing solution is applied to a glass coverslip which is then inverted so as to suspended the droplet above a larger reservoir of a similar solution lacking protein but containing a higher concentration of precipitant, and the chamber sealed. Over time, the droplet containing protein equilibrates with the larger reservoir beneath it as volatile water in the droplet leaves the droplet and transfers to the reservoir, effectively increasing the precipitant concentration in the protein droplet. In solutions of a favourable composition, the protein becomes supersaturated and crystal nuclei form, leading to crystal growth. That is the optimal outcome. Otherwise (and typically) the protein forms a useless and amorphous mass as protein precipitates out of solution. 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 angstroms (400 picometers). Many biomolecules of interest still have not been successfully crystallised. Imperfections in the crystal structure, caused by impurities, sample contamination, or multiple stable conformations of the subject protein 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 then mounted. Several methods of mounting exist, it is possible to hold the crystal in a thin glass tube using grease or by using superglue or epoxy resin to hold the crystal to a glass fibre. A modern alternative is to use a drop of oil and liquid nitrogen to fix the crystal to the fibre. By cooling crystals the radiation damage incurred during data collection is reduced 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 sealed X-ray tube with a stationary anode (circa 2 kW DC input), a rotating-anode type source (circa 14 kW DC input) or a synchrotron (much higher photon flux). 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. Before the advent of cryocooling, all data was collected at room temperature: Increased radiation damage to the crystal meant that sometimes several crystals had to be used to obtain a single dataset - cryocooling has reduced this problem. Moreover, instead of collecting the data spots one at a time, many modern machines use an array of X-ray detectors (a CCD array) to collect data over a large range of angles at once. Data processing
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