Computer Assisted Modeling Contributions of Computational Approaches to Elucidating Macromolecular Structure and Function (1987) / Chapter Skim
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5 Tertiary Structure of Proteins and Nucleic Acids: Experimental
5 Tertiary Structure of Proteins and Nucleic Acids: Experimental
Pages 41-68
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From page 41... ...
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.
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From page 42... ...
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.
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From page 43... ...
Consequently, we can now use the known structure to obtain phase information about an unknown structure, a method known as molecular replacement (Rossmann, 1972~. There have been numerous examples of the successful application of molecular replacement to determine crystal structures.
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From page 44... ...
But in the area of macromolecular structural analysis, particularly with DNA and its complexes with antitumor drugs and control proteins such as repressors, computer graphics will illuminate by enabling the investigator to carry out the structure analysis efficiently and to see aspects of the structure of the molecule that he could perceive only with difficulty or would overlook entirely using more traditional methods. In the early 1950s, before any protein structure had been determined by x-ray methods, the British crystallographer J
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From page 45... ...
being constructed (Figure 5-1~. In the 1960s and 1970s, virtually all macromolecular structure groups had at least one Richards Box to use in interpreting electron density maps and building macromolecular models into them.
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From page 46... ...
45: F1~UiE ~5~1~ ll~l~st~^t~ ~ the S~>thSS (~/ I the ~ fief ~ipS{6r gamy ~ ~te~t~el~t=~ ages; If ~ =~1~1~ S~ ~ < bag. S=t~s twang)
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From page 47... ...
. ~ ~ tron density In one ot several options: a simple Fo electron density map, a (Fo ~ Fc)
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From page 48... ...
Computer graphics per se is of relatively little assistance in the subsequent process of refinement of heavy atoms and single or multiple ~somorphous replacement phase analysis. But once a rough electron density map of the DNA helix is calculated, graphics again proves extremely useful.
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From page 49... ...
In this particular application, the conventional minimap was tedious, but the graphics display was very simple to interpret. Location of Solvent Molecules and ions Around a Macromolecule The images of solvent molecules around a macromolecule cannot all be found from unrefined electron density maps.
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From page 50... ...
The framework is a representation in three dimensions of one contour level in the electron density of the drug, and the graphics operator has built a skeleton of the netropsin molecule within this contour cage. This is the first point at which information about the drug was built into the analysis and was the point of departure for further least-squares refinement of the DNA-drug complex.
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From page 51... ...
Such a search for solvent requires the inspection of many successive electron density maps as refinement proceeds, and computer graphics are of enormous help in speeding up this process.
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From page 52... ...
In this view, the bases of each individual strand of the double helix are seen to be stacked atop one another, almost as though the other strand of the helix did not exist. This efficiency of intrachain base stacking means that when the two strands are wound around one another to build a double helix, the bases of each base pair are not coplanar; they are given what is defined as a positive propeller twist about the Tong axis connecting them.
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From page 53... ...
Examples of this serendipity with computer graphics are found over and over again in macromolecular structure analysis.
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From page 54... ...
The figure illustrates the structurally significant finding that the ends of the molecule are more closely associated with the DNA than is the central amide. One can change base pairs in the DNA from AT to 2-aminoadeninethymine and show the steric clash that then ensues with the drug.
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From page 55... ...
55 -a Ha> Hi< rip at: _.< a_ Am< FIGURE 5-4 Oblique computer graphics stereo diagram of the complex of netropsin with C-G-C-G-A-A-T-T-C-G-C-G, in a view sighting directly down the minor groove with the drug molecule slotted into it in a crescent curving away from the viewer. This view also illustrates the strong stacking of bases down each strand of a Beta-DNA double helix, in a more striking representation than is obtained from a more conventional view of the Beta helix.
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From page 56... ...
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From page 57... ...
However, several groups have determined peptide or protein structure for molecules in the 5 to 10 kDa molecular weight range (Arseniev et al., 1984; Braun et al., 1986; Have} and Wuthrich, 1985; Kaptein et al., 1985; Kline et al., 1986; Williamson et al., 1985~. A brief discussion of the protein examples provides insight into the potential contribution NMR may make to the prediction of macromolecular structure and function in years to come.
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From page 58... ...
Resolution in conventional spectral acquisitions is inadequate to identify each resonance, let alone assign it to a primary structure site or acquire NOE data on a significant fraction of the possible 5 x 105 proton pairs. The situation with other types of macromolecules, nucleic acids or oligosaccharides, is less formidable in terms of numbers of protons.
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From page 59... ...
To determine a structure, a limited amount of longer range distance constraint information must be systematically integrated with other constraints. The most direct approach probably employs a distance geometry search for structures that have interproton distances between the experimentally determined upper and lower bounds (Braun and Go, 1985; Havel et al., 1979~.
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From page 60... ...
deviations of heavy atoms in multiple structure solutions of proteins obtained from NMR data suggest resolution to be approxanately 3 ~ (Williamson et al., 1985~. Although these average deviations are larger than those usually seen in x-ray data, NMR methods can be used in a variety of media and yield very precise distance information on selected distances.
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From page 61... ...
Replacement of normal amino acids with amino acids that contain i5N and i3C and the use of indirect detection methods to improve sensitivity also allow use of the increased chemical shift dispersion displayed in spectra of other nuclei. Proteins of 19 kDa (McIntosh et al., 1987)
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From page 62... ...
A rather limited quantity of residue-specific information may be required to improve dramaticaDy the quality of theoretical predictions. A second limitation, to current methodology beyond accessible macromolecule size, stems from the restricted range of measurable interproton distances, <41.
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From page 63... ...
One may also ask whether studies of small protein or peptide structure might be relevant for the understanding of larger structures. Although some behavior, such as allosteric interaction, is certain to be poorly represented in small molecules, the basic structural considerations are reasonably likely to carry over, and it is likely that fundamental processes such as protein folding can be studied.
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From page 64... ...
The same two-dimensional NMR methods applied to proteins allows us to characterize nucleic acid structures and explore in detail the factors that lead to interconversion of structural forms. The imino protons involved in the hydrogen bond connecting base pairs exchange slowly with protons in the solvent and are easily resolved in the low field region of a proton NMR spectrum.
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From page 65... ...
More quantitative treatments of structure employ the same molecular mechanics, molecular dynamics, and distance geometry methods used with proteins (Hare and Reid, 1986; Nilsson et al., 1986; Suzuki et al., 1986~. Beyond simple helical structures lies a vast region of structural biology of nucleic acids that has been much less explored.
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From page 66... ...
DEMAND ON COMPUTATIONAL FACILITIES Improved computational and molecular modeling facilities could advance structure determination using NMR methods in several ways. Processing and analysis of NMR data for a 10 kDa macromolecule is far more time-consuming than is data acquisition.
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From page 67... ...
Several approaches are being explored to reduce these complex peaks to a few pieces of connectivity information, but it is possible that methods such as linear decomposition, which reduce frequency domain sets to lists of peak frequency and intensity at an early stage, will provide the breakthrough needed in this area. Once resonances have been cataloged, the next step is the assignment and extraction of spectral characteristics important for the determination of secondary and tertiary structures.
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From page 68... ...
In summary, current NMR methods to determine structure seem applicable to a variety of biologically unportant molecules of less than 10 kDa. Data production in this clam will be made much easier by improved computational facilities, available high field spectrometers, and attention to the compatibility of modeling programs with experimental constraints provided by NMR.
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