Protein crystallography isomorphous replacement

In small-molecule crystallography the phase problem was solved by so-called direct methods (recognized by the award of a Nobel Prize in chemistry to Jerome Karle, US Naval Research Laboratory, Washington, DC, and Herbert Hauptman, the Medical Foundation, Buffalo). For larger molecules, protein aystallographers have stayed at the laboratory bench using a method pioneered by Max Perutz and John Kendrew and their co-workers to circumvent the phase problem. This method, called multiple isomorphous replacement... 

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. 

The problem of phase determination is the fundamental one in any crystal structure analysis. Classically protein crystallography has depended on the method of multiple isomorphous replacement (MIR) in structure determination. However lack of strict isomorphism between the native and derivative crystals and the existence of multiple or disordered sites limit the resolution to which useful phases may be calculated. 

The classical method for solving the phase problem in macromolecular crystal structures, known as isomorphous replacement, dates back to the earliest days of protein crystallography.10,16 The concept is simple enough we introduce into the protein crystal an atom or atoms heavy enough to affect the diffraction pattern measurably. We aim to figure out first where those atoms are (the heavy atom substructure) by subtracting away the protein component, and then bootstrap — use the phases based on the heavy atom substructure to solve — the structure of the protein. 

However, when the intensities of the X-rays are recorded in this manner all information of the phase is lost. Thus, the fundamental problem in a structure determination is the phase problem. Until recently, the phase problem in protein crystallography has been solved by the heavy atom isomorphous replacement method (sections 2(d) and (e)), but other methods are also available (sections 2(e) and (f)). 

In protein crystallography, the technique of isomorphous replacement is used to ... 

Tel. 44-925-603528, fax 44-925-603100, e-mail ccp4 daresbury.ac.uk Suite of almost 100 protein crystallography programs for data processing, scaling, Patterson search and refinement, isomorphous and molecular replacement, structure refinement, such as PROLSQ, phase improvement (solvent flattening and symmetry averaging), and presentation of results, such as SURFACE for accessible surface area. Available via ftp from anonymous ... 

These authors describe the use of SIROAS applied to protein crystallography in general, and to glutamate dehydrogenase in particular involving a mercury derivative. The acronym, SIROAS, is an adaptation of the standard Cu Ka acronym SIRAS, single isomorphous replacement... 

Some of the so-called physical methods are among the most commonly applied in protein crystallography as the isomorphous replacement method and the anomalous scattering method. 

In early 1948 I thought that there was an experimental solution of the phase problem of X-ray crystallography. The idea was to use a double reflection hj followed by I12 which diffracts in the direction of I13 = hi + I12. If hi is set on the sphere of reflection so that it diffracts for any orientation of the crystal about a suitably chosen rotation axis, then hi and I12 should show an interference effect. This idea, beautiful in principle, was defeated by the mosaic character of crystals and possibly also crystal boimdary effects. Our experiment in which hi is 040 of a glycine crystal failed, although some reflections which were forbidden as single diffractions were observed. Shortly thereafter (1951) Bijvoet published his experimental solution to the phase problem by multiple isomorphous replacement methods, and I thought then that his discovery opened the way to solve protein structures. However, I did not start work in this direction until about 1958, and pursued it seriously beginning in 1961. 

Thermolysin (m.w. 37 500) has 4 binding sites for Ca denoted for convenience as Cl, C2, C3, and C4. It was with this protein that Colman et al. (1972) demonstrated the potential of the lanthanides to serve as isomorphous replacements for Ca " in protein crystallography. The system has subsequently been studied in greater detail by Matthews and Weaver (1974). It was found that R " may occupy sites Cl, C3, and C4 but not C2 which is only 3.8 A away from Cl. In fact the Ca ions at Cl and C2 share some of the carboxyl side chains forming these sites. There is little perturbation of protein structure upon lanthanide substitution, e.g. the sites occupied by Eu ions differ by only 0.3-0.7 A from those of the displaced Ca ions. Site Cl is composed of the carboxyl side chains of Asp-185, Glu-177, and Glu-190. Site C3 includes the carboxyl side chains of Asp-57 and Asp-59. There is only one carboxyl, that of Asp-200, at C4. Substitution experiments carried out with crystals have shown... 

For macromolecules such as proteins, the numbers of atoms that compose molecules are huge, therefore the crystal cells contain large numbers of atoms. It is not possible to apply the methods for small molecules, such as the direct method or Patterson map searching, in the structure determinations of proteins. The methods for retrieving the phases of protein crystal diffractions are molecular replacement, isomorphous replacement and anomalous scattering. In recent years, the direct method, which has been widely and successfully used in the determination of small-molecule structures, has also been applied in protein crystallography. 

Isomorphous replacement is the keystone of protein crystallography, by which the first protein structure was solved. This is also the first method to... 

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. 

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