Crystallography isomorphous replacement methods

PEPC from E. coli was crystallized as described previously [1] with minor modifications. X-ray diffraction data were collected at station BL-6B of the Photon Factory, Japan. Intensity data were obtained using a Weisssenberg camera for macromolecule crystallography and imaging plates as a detector [5]. The data were processed using DENZO and scaled by the program SCALEPACK [6]. The crystal structure was determined by multiple isomorphous replacement method. 

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)). 

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. 

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 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. 

V is the volume of the unit cell and should not be confused with V(r) = V(xyz). From the fact that only the absolute square of F can be measured, the phase of the complex value of F is lost. This is the phase problem of crystallography, which found its solution by the introduction of the method of isomorphous replacement in the fifties... 

Deisenhofer was independent, too. This project was not simple crystallography, not at all. It was a most complex structure determination. Even the methods of measuring the intensities were not automated at the time. We had developed instruments. X-ray cameras and methods for that purpose also. We had a small workshop at that time, with a mechanic and an electronics person. One day the mechanic had a stroke. A week later the electronics person had a heart attack. They were very important in servicing the instruments. I was the only one in my department able to service the instruments. It was at a critical time for the photosynthetic reaction center work, around 1983, and I spent much time each day taking care of the instruments. It is a side issue, not even a scientific one, but it shows you that things may look different from the perspective of today s possibilities than they actually were. The work at that time required a background also concerning the availability of the samples for isomorphous replacement and methods to apply them. I have made many of these samples and I built up an enormous collection. 

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. 

X-ray crystallography is currently the most powerful analytical method by which three-dimensional structure information on biological macromolecules may be obtained at high resolution. Its application is however limited first by the preparation of single crystals suitable for X-ray diffraction and second by the so-called phase problem , that is the calculation of phases of difBaction data. Several approaches are available in order to circumvent this latter problem. The most commonly used methods are the multiple and single isomorphous replacement (MIR, SIR). These methods, as well as multiple anomalous difBaction (MAD), require the preparation of heavy atom derivatives, usually by the introduction of electron-dense atoms at distinct locations of the crystal lattice. This is usually done by crystal soaking experiments. 

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... 

---