How to identify tertiary structure afterproteinpurification?
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X-RAY DIFFRACTION OF BIOLOGICAL MACROMOLECULES
In 1934 Bernal and Crowfoot demonstrated that a crystalline protein could give rise to a well-ordered x-ray diffraction pattern, thus setting the stage for modern analysis of the structure of proteins. Progress was gradual at first, interrupted by World War II, but in 1953 Green, Ingram, and Perutz took another essential step when they accomplished the first heavy atom analysis of a hemoglobin crystal (Green et al., 1954). The culmination of these years of work came in 1959 when Kendrew and his colleagues (1960) reported the analysis of myoglobin at 2 Å resolution, revealing for the first time the underlying structure of a globular protein. They noted the complexity and lack of regularity of the molecule—major features that continue to impress us today as general features of protein structure. The alpha-helices and beta sheets of Pauling and Corey (Pauling et al., 1951) form striking regions of regularity, but are joined together in very complex ways.
Another major event of protein structure analysis occurred the same year when Cullis et al. (1959) described the structure of hemoglobin at 6 Å and demonstrated that the folding of the globin chain is similar to that in myoglobin, despite relatively low sequence homology between the two. This observation of a family pattern to the three-dimensional structure of globins has been followed by the identification of many other families.
Today, several hundred proteins have been analyzed by x-ray diffraction and their three-dimensional structures catalogued. This number continues to grow at an ever-increasing rate and, together with the amino acid and gene sequence data, forms the principal basis for understanding the mechanisms of action of these proteins at the molecular level.
The development of two-dimensional nuclear magnetic resonance (NMR) techniques already is a valuable complement to x-ray diffraction for relatively small molecules (less than 10,000 molecular weight) but for the foreseeable future, crystal structure analysis will be the principal experimental source of structural data for enzymes, nucleic acid binding proteins, antibodies, and other proteins of the immune system, receptors, and indeed, for all proteins that can be effectively crystallized.
Determining the three-dimensional structure of a biological macromolecule by crystallography involves a number of clearly defined steps. First, crystals of suitable size and diffraction properties must be prepared. Next, x-ray diffraction data must be collected for these crystals and also, typically, for a number of heavy atom derivatives of the crystals. These data can then be assembled to obtain an electron density map using a computational process that resembles the action of the lens in a microscope. This map must then be fitted with a polypeptide chain of the appropriate amino acid sequence. Because the map is of less-than-atomic resolution and also contains errors in the phase determination, the investigator must have considerable skill to obtain the best fit. The resulting protein model must then be refined to remove the errors present in the map as much as possible as well as those errors introduced by the fitting process.
Computers play an essential role in most of these steps. Even the analyses of the first protein crystal structure, myoglobin, could not have been accomplished without the use of the EDSACII in Cambridge (Kendrew, 1960). At present, modern crystallography depends completely on heavy computer use, and this dependence will certainly increase steadily in the future. In the four mathematical procedures required to solve a structure using protein crystallography—data processing, phase determination, map fitting, and refinement—new methods are continually appearing that depend on ready access to considerable computer power.
In 1934 Bernal and Crowfoot demonstrated that a crystalline protein could give rise to a well-ordered x-ray diffraction pattern, thus setting the stage for modern analysis of the structure of proteins. Progress was gradual at first, interrupted by World War II, but in 1953 Green, Ingram, and Perutz took another essential step when they accomplished the first heavy atom analysis of a hemoglobin crystal (Green et al., 1954). The culmination of these years of work came in 1959 when Kendrew and his colleagues (1960) reported the analysis of myoglobin at 2 Å resolution, revealing for the first time the underlying structure of a globular protein. They noted the complexity and lack of regularity of the molecule—major features that continue to impress us today as general features of protein structure. The alpha-helices and beta sheets of Pauling and Corey (Pauling et al., 1951) form striking regions of regularity, but are joined together in very complex ways.
Another major event of protein structure analysis occurred the same year when Cullis et al. (1959) described the structure of hemoglobin at 6 Å and demonstrated that the folding of the globin chain is similar to that in myoglobin, despite relatively low sequence homology between the two. This observation of a family pattern to the three-dimensional structure of globins has been followed by the identification of many other families.
Today, several hundred proteins have been analyzed by x-ray diffraction and their three-dimensional structures catalogued. This number continues to grow at an ever-increasing rate and, together with the amino acid and gene sequence data, forms the principal basis for understanding the mechanisms of action of these proteins at the molecular level.
The development of two-dimensional nuclear magnetic resonance (NMR) techniques already is a valuable complement to x-ray diffraction for relatively small molecules (less than 10,000 molecular weight) but for the foreseeable future, crystal structure analysis will be the principal experimental source of structural data for enzymes, nucleic acid binding proteins, antibodies, and other proteins of the immune system, receptors, and indeed, for all proteins that can be effectively crystallized.
Determining the three-dimensional structure of a biological macromolecule by crystallography involves a number of clearly defined steps. First, crystals of suitable size and diffraction properties must be prepared. Next, x-ray diffraction data must be collected for these crystals and also, typically, for a number of heavy atom derivatives of the crystals. These data can then be assembled to obtain an electron density map using a computational process that resembles the action of the lens in a microscope. This map must then be fitted with a polypeptide chain of the appropriate amino acid sequence. Because the map is of less-than-atomic resolution and also contains errors in the phase determination, the investigator must have considerable skill to obtain the best fit. The resulting protein model must then be refined to remove the errors present in the map as much as possible as well as those errors introduced by the fitting process.
Computers play an essential role in most of these steps. Even the analyses of the first protein crystal structure, myoglobin, could not have been accomplished without the use of the EDSACII in Cambridge (Kendrew, 1960). At present, modern crystallography depends completely on heavy computer use, and this dependence will certainly increase steadily in the future. In the four mathematical procedures required to solve a structure using protein crystallography—data processing, phase determination, map fitting, and refinement—new methods are continually appearing that depend on ready access to considerable computer power.
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