Isomorphous heavy atom

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

The metallointercalation reagents are a class of heavy metal derivatives that bind to double-stranded polynucleotides by inserting between adjacent base pairs in the helix.1 2 Prototype members of this class of intercalators are (2,2 6, 2"-terpyridine)(thiolato)platinum(II) complexes.3 These may be synthesized from chloro(2,2 6, 2"- terpyridine)platinum(II), which can both intercalate and bind covalently by losing chloride ion. Covalent binding of the thiolato complexes is much slower owing to the more inert character of the Pt—S bond. Metallointercalation reagents also have the potential to bind to proteins that have natural receptor sites for nucleic acid bases. They may therefore also be used to provide isomorphous heavy atom derivatives for X-ray analysis. 

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. 

FIGURE 8.3 A hypothetical series of isomorphous heavy atom derivatives for a crystalline macromolecule, represented here by the polypeptide backbone of rubredoxin. (a) The apo-protein, stripped of its metal ion, provides native structure factors />, shown in vector and waveform on the right (b) the protein with its naturally bound iron atom and FHi, the first derivative structure factor (c) the protein with its iron plus an attached mercury atom, and the resultant structure factor Fm from the double derivative (d) a second multiply substituted derivative formed by attachment of a gold atom to the protein-iron complex. This last derivative is only marginally useful, however, since the reaction with gold also produces a modification in the tertiary structure of the protein (denoted by an arrow). Since this non-isomorphism is equivalent to introducing a nonnative structure factor contribution, the observed F s cannot be properly accounted for, and an erroneous heavy atom contribution / results. This final derivative will yield an inaccurate phase estimate 0v for the native protein. 

FIGURE 8.6 For any native phase angle p the vector triangle T ru = F/> + fn fails to close by an amount e(p). which is termed the lack of closure error. The phase angle of maximum probability, that for which e(p). is minimized when considered over all isomorphous heavy atom derivatives. 

FIGURE 8.7 The top graph shows the separate probabilities over the entire range 0 to 1 for two isomorphous heavy atom derivatives I and II. Both are bimodal and each separately predicts the two most likely phase angles for the native structure factor. At the bottom the joint probability distribution strongly predicts a single most probable phase. 

For most proteins, the phase information is obtained from isomorphous heavy atom derivatives. A heavy atom (e.g., Hg, U or Pt), introduced into the protein, scatters more than the light atoms (C,N,0) of the protein and is used as a marker atom. 

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... 

Figure 6.26 Representation in the Gaussian plane of the phase relationships between the structure factor of a pure protein, FP, a heavy atom, FH and an isomorphic heavy atom derivative of the protein,...

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

The more automatic solution of crystal structures using variable or multiple wavelength anomalous scattering measurements is an important development especially when conventional, isomorphous, heavy atom derivatives cannot be made. The phasing power of the anomalous scattering component of specific atoms can be fully optimised with SR. 

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. 

Only one of the crystallized proteins has yet matured into a crystal structure. This is the L7/L12 CTF (Leijonmarck et al., 1980 Leijonmarck and Liljas, 1982). The structure was initially solved with phase angles determined from isomorphous heavy atom derivatives at 2.6 % resolution. Subsequently the structure has been refined and the phase angles extended stepwise to 1.7 % resolution. 

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