Isomorphous heavy atom replacement

The analysis of water structure can be further enhanced by mixing H2O and D2O which like isomorphous heavy atom replacement,... 

The most general method of solving the phase problem for protein crystals is that of multiple isomorphous replacement in which two or more isomorphous heavy-atom derivatives are used.1 The principle of the method is shown in Figure 3. In Figure 3a a circle with radius Fp, the amplitude of a reflection from the native protein, is shown with center at the origin, O. It is assumed that the heavy atoms in at least two derivatives have been located and referred to the same unit cell origin. This can be a difficult problem and mistakes can be made, but... 

Preparation of isomorphous heavy-atom derivatives. The multiple isomorphous replacement technique has been commonly used to solve biomacromolecular structures. It requires the parent crystal and at least two heavy-atom derivatized crystals identical in space group and molecular structure. The common technique uses reagents containing heavy atoms and allows them to diffuse into the crystal. 

The preparation of isomorphous heavy-atom derivatives. If only one iso-morphous derivative is prepared, the method is known as a single isomorphous replacement if more than one derivative is prepared the method is known as multiple isomorphous replacement. 

MIR), requires the introduction of new x-ray scatterers into the unit cell of the crystal. These additions should be heavy atoms (so that they make a significant contribution to the diffraction pattern) there should not be too many of them (so that their positions can be located) and they should not change the structure of the molecule or of the crystal cell—in other words, the crystals should be isomorphous. In practice, isomorphous replacement is usually done by diffusing different heavy-metal complexes into the channels of preformed protein crystals. With luck the protein molecules expose side chains in these solvent channels, such as SH groups, that are able to bind heavy metals. It is also possible to replace endogenous light metals in metal-loproteins with heavier ones, e.g., zinc by mercury or calcium by samarium. 

X-Ray diffraction from single crystals is the most direct and powerful experimental tool available to determine molecular structures and intermolecular interactions at atomic resolution. Monochromatic CuKa radiation of wavelength (X) 1.5418 A is commonly used to collect the X-ray intensities diffracted by the electrons in the crystal. The structure amplitudes, whose squares are the intensities of the reflections, coupled with their appropriate phases, are the basic ingredients to locate atomic positions. Because phases cannot be experimentally recorded, the phase problem has to be resolved by one of the well-known techniques the heavy-atom method, the direct method, anomalous dispersion, and isomorphous replacement.1 Once approximate phases of some strong reflections are obtained, the electron-density maps computed by Fourier summation, which requires both amplitudes and phases, lead to a partial solution of the crystal structure. Phases based on this initial structure can be used to include previously omitted reflections so that in a couple of trials, the entire structure is traced at a high resolution. Difference Fourier maps at this stage are helpful to locate ions and solvent molecules. Subsequent refinement of the crystal structure by well-known least-squares methods ensures reliable atomic coordinates and thermal parameters. 

The structure was solved by the multiple isomorphous replacement technique using four heavy atom derivatives uranyl acetate, plati-nous chloride, tetramethyllead acetate, and p-chloromercury benzoate. All four derivatives gave interpretable heavy atom Patterson syntheses. The heavy atom sites could be correlated between the de-... 

Multiple isomorphous replacement allows the ab initio determination of the phases for a new protein structure. Diffraction data are collected for crystals soaked with different heavy atoms. The scattering from these atoms dominates the diffraction pattern, and a direct calculation of the relative position of the heavy atoms is possible by a direct method known as the Patterson synthesis. If a number of heavy atom derivatives are available, and... 

In the elucidation of the X-ray structure of hCP by the method of isomorphous replacement, gold and mercury heavy atom derivatives were utilized. In the case of the mercury derivative, p-chloromercury-benzoate, the heavy atom bound to the free sulphydryl residue, C221, but for the gold cyanide derivative the gold atom was found to bind in the vicinity of the trinuclear copper cluster. A realistic explanation of this... 

The isomorphous replacement method requires attachment of heavy atoms to protein molecules in the crystal. In this method, atoms of high atomic number are... 

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

Isomorphous Replacement (Heavy Atom/Metal Derivatives). 464... 

Isomorphous replacement Heavy atoms (atoms with high atomic numbers, often metals) are attached to the protein molecules. 

The isomorphous replacement method requires attachment of heavy atoms to protein molecules in the crystal. In this method, atoms of high atomic number are attached to the protein, and the coordinates of these heavy atoms in the unit cell are determined. The X-ray diffraction pattern of both the native protein and its heavy atom derivative(s) are determined. Application of the so-called Patterson function determines the heavy atom coordinates. Following the refinement of heavy atom parameters, the calculation of protein phase angles proceeds. In the final step the electron density of the protein is calculated. 

Figure 6.3 Isomorphous replacement phase determination (Marker construction), (a) Single isomorphous replacement. The circle with radius Fpp represents the heavy-atom derivative, while that with radius Fp represents the native protein. Note that the circles intersect at two points causing an ambiguity in the phase angle apg and apt, represent the two possible values, (b) Double isomorphous replacement. The same construction as that in single isomorphous replacement except that an additional circle with radius Fpn2 (vector not shown for simplicity) has been added to represent a second heavy-atom derivative. Note that all three circles (in the absence of errors) intersect at one point thus eliminating the ambiguity in the protein phase angle ap. Fm and Ppy represent the heavy-atom vectors for their respective derivatives.

La Fortelle, E. D. and Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Method Enzymol. 276, 472 94. 

In most cases, however, the protein molecules are larger or the resolution of the data is lower, and phasing becomes a two-stage process. If two or more intensity measurements are available for each reflection with differences arising only from some property of a small substructure, the positions of the substructure atoms can be found first, and then the substructure can serve as a bootstrap to initiate the phasing of the complete structure. Suitable substructures may consist of heavy atoms soaked into a crystal in an isomorphous replacement experiment, or they may consist of the set of atoms that exhibit... 

In order to understand the interactions between these bis-intercalating drugs and IMA more fully, we have crystallized several complexes of them and undertaken the structure determination by x-ray diffraction technique. One of the crystal forms diffracts to 1.6 A resolution with a space group of F222. The crystal structure was determined by the multiple isomorphous replacement method using three different heavy atom derivatives. The structure was refined to an R-factor of 19% and there were moderate number of solvent molecules clearly visible. The crystal... 

In crystallography, heavy atom derivatives are required to solve the phase problem before electron density maps can be obtained from the diffraction patterns. In nmr, paramagnetic probes are required to provide structural parameters from the nmr spectrum. In other forms of spectroscopy a metal atom itself is often studied. Now many proteins contain metal atoms, but even these metal atoms may not be suitable for crystallographic or spectroscopic purposes. Thus isomorphous substitution has become of major importance in the study of proteins. Isomorphous substitution refers to the replacement of a given metal atom by another metal that has more convenient properties for physical study, or to the insertion of a series of metal atoms into a protein that in its natural state does not contain a metal. In each case it is hoped that the substitution is such that the structural and/or chemical properties are not significantly perturbed. 

A knowledge of the structure of proteins has long been recognized as fundamental to an understanding of their chemical and biological functions. One technique for the successful determination of the X-ray crystal structure of proteins was initiated by Perutz in 1954 and involved the isomorphous replacement of heavy atoms into the protein crystal.412 The development of this technique was recently reviewed.413... 

The isomorphous replacement method becomes ineffective for protein crystals beyond a resolution of 2 to 2.5 A, because the addition of a heavy atom may cause some alteration in structure and it is difficult to observe small changes in intensities that are already very weak. But, structures may be refined to high resolution, as in the molecular replacement procedure, by using the measured intensities at higher resolution and the model structure that has been determined at resolutions of 2 to 2.5 A. 

The most demanding element of macromolecular crystallography (except, perhaps, for dealing with macromolecules that resist crystallization) is the so-called phase problem, that of determining the phase angle ahkl for each reflection. In the remainder of this chapter, I will discuss some of the common methods for overcoming this obstacle. These include the heavy-atom method (also called isomorphous replacement), anomalous scattering (also called anomalous dispersion), and molecular replacement. Each of these techniques yield only estimates of phases, which must be improved before an interpretable electron-density map can be obtained. In addition, these techniques usually yield estimates for a limited number of the phases, so phase determination must be extended to include as many reflections as possible. In Chapter 7,1 will discuss methods of phase improvement and phase extension, which ultimately result in accurate phases and an interpretable electron-density map. 

Several diffraction criteria define a promising heavy-atom derivative. First, the derivative crystals must be isomorphic with native crystals. At the molecular level, this means that the heavy atom must not disturb crystal packing or the conformation of the protein. Unit-cell dimensions are quite sensitive to such disturbances, so heavy-atom derivatives whose unit-cell dimensions are the same as native crystals are probably isomorphous. The term isomorphous replacement comes from this criterion. 

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