Isomorphous heavy-atom derivatives, preparation

Although the soaking method for heavy-atom derivative preparation is by far the simplest and most common, it is not the only method used. One can first derivatize the macromolecule, and then crystallize. This procedure is less frequently used because of drawbacks such as the inabihty to produce isomorphous crystals due to the disruption of intermolecular contacts by the heavy atoms. Other frequent problems are the introduction of additional heavy-atom sites (a potential complicating factor in phasing) by exposing sites hidden by crystal contacts, and changing the solubility of the derivatized macromolecule. 

Petsko, G. A. (1985). Preparation of isomorphous heavy-atom derivatives. Method Enzymol. 114,147-156. 

A number of studies have been performed with methyl picolinimidate (Benisek and Richards 1968 Plapp et al. 1971) aimed at exploring the usefulness of the metal-chelating properties of such derivatives in the preparation of isomorphous heavy atom derivatives of proteins for X-ray diffraction studies. 

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. 

Heavy-atom derivation of an object as large as a ribosomal particle requires the use of extremely dense and ultraheavy compounds. Examples of such compounds are a) tetrakis(acetoxy-mercuri)methane (TAMM) which was the key heavy atom derivative in the structure determination of nucleosomes and the membrane reaction center and b) an undecagold cluster in which the gold core has a diameter of 8.2 A (Fig. 14 and in and ). Several variations of this cluster, modified with different ligands, have been prepared The cluster compounds, in which all the moieties R (Fig. 14) are amine or alcohol, are soluble in the crystallization solution of SOS subunits from H. marismortui. Thus, they could be used for soaking. Crystallographic data (to 18 A resolution) show isomorphous unit cell constants with observable differences in the intensity (Fig. 15). 

X 77.8 X 51.4 A, a space group of P2i2i2j, and a Z value of 4 (molecules per unit cell) (46). Several heavy atom derivatives, whose crystalline structure are isomorphous with that of the native enzyme, have been prepared for the X-ray diffraction analysis of crystalline cytochrome c peroxidase. 

Heavy atom derivatives of a macromolecular crystal can be prepared (Green, Ingram and Perutz 1954) which for a minimum of two derivatives (and the native crystal) and in the absence of errors, leads to a unique determination of the phase ahkt in equation (2.7) (figure 2.13(a)). This requires the site and occupancy of the heavy atom to be known for the calculation of the vector FH (the heavy atom structure factor). In the absence of any starting phase information the heavy atom is located using an isomorphous difference Patterson synthesis P(u,v,w) where the isomorphous difference is given by... 

Calculation of an electron density map. Several heavy-atom derivatives are prepared and isomorphous replacement has been used to estimate phases for aU Fp(h, k, 1). These phases are used to calculate an electron density map of the crystal and aU data to a certain resolution. 

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. 

We also have crystals of chymosin (6), the acid proteinase from Mucor pusillus (7), chicken pepsin (8), and chicken pepsinogen (8), the first two of which are large and very suitable for x-ray analysis. Dr. C. W. Bunn and his co-workers made preliminary x-ray studies of chymosin but the method of isomorphous replacement was unsuccessful, as simple heavy atom derivatives proved impossible to prepare. Both we and Professor B. Foltmann and Dr. S. Larsen of Copenhagen have continued x-ray studies on chymosin, but have also met difficulties. More recently, we have began to use the structural information from the Endothia parastica enzyme to solve the structure of chymosin by using the method of molecular replacement. 

The fourth step is the preparation of isomorphous crystals of heavy metal-containing derivatives. The heavy metal may be allowed to react with the protein before crystallization or may be diffused into preformed crystals. A variety of both cationic and anionic metal complexes, even large Ta6Br122+ tantalum clusters, have been used 405 Two or more different heavy metal derivatives are often required for calculation of the phases. The heavy metal atoms must be present at only a very small number of locations in the unit cell. 

Crystals of the material are grown, and isomorphous derivatives are prepared. (The derivatives differ from the parent structure by the addition of a small number of heavy atoms at fixed positions in each — or at least most — unit cells. The size and shape of the unit cells of the parent crystal and the derivatives must be the same, and the derivatization must not appreciably disturb the structure of the protein.) The relationship between the X-ray diffraction patterns of the native crystal and its derivatives provides information used to solve the phase problem. 

The chemical preparation step in which the protein is isolated, purified, and crystallized is critical in that the protein preparation must be chemically homogeneous otherwise, the resulting disorder will muddle the electron-density map. The preparation of isomorphous derivatives by soaking native protein crystals in various mercury, platinum, lead, uranium, etc., solutions also is critical since several crystals of each derivative are required for x-ray data collection (because of irradiation damage) and all the crystals should have the same heavy-atom distribution and concentration. The protein structure documentation should provide evidence that the preparative protein chemistry is sound. 

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