Method for obtaining protein crystals
Protein structural information for the inhibited complexes F1-I1-60His and F1-resveratrol were obtained by Protein Crystallography. Protein crystallography is a method that allows scientists to determine the three-dimentional molecular structures of crystallized proteins and other large molecules. The three dimensional structures that are predicted from x-ray analysis show the primary, secondary, tertiary, and quaternary structures of proteins. Protein crystallography is a meticulous and difficult process that involves numerous steps (Figure 8).
Figure 8: The major steps of crystallography; crystallization, x-ray analysis into a diffraction pattern, comparison to an electron-density map, and fitting to an atomic model. Proxy Chem
Crystallization Step 1: Purification
The first an often most difficult phase of the crystallization is to purify the protein. It is very important that the protein is extremely pure (>97%) because proteins can be very fragile, easily losing activity during the process. Structures of many important proteins remain a mystery simply because researchers are unable to obtain adequate crystals due to impurities (Guide to Protein Crystallography).
Crystallization Step 2: Cultivate Crystals
Once high purity is attained, protein crystallization can be attempted. Due to the inherent fragility of proteins, important factors such as the pH, concentration, and temperature must be considered when attempting to crystallize them. PH conditions can be critically important because different pH's can result in different packing orientations (Guide to Protein Crystallography). In order to crystallize a purified protein, it must undergo slow precipitation from solution such that the individual protein molecules align themselves in a repeating series of 'unit cells'. As the unit cells adopt a consistent orientation, the crystals are held together by non covalent interactions (Rhodes 1993).
Crystallization Step 3:
After sufficient-sized crystals have been obtained (>0.1 mm per side), they are mounted and coordinated in an x-ray detector. Then an x-ray beam is diffracted into a pattern that are analyzed to produce a three dimensional map of the molecule's electron density. The data is processed by fitting a model of the protein's known sequence to the diffraction pattern. the protein structure can then be solved, refine and analyzed (Guide to Protein Crystallography).
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