Heavy-atom Derivatives

Referring to figure Bl.8.5 the radii of the tliree circles are the magnitudes of the observed structure amplitudes of a reflection from the native protein, and of the same reflection from two heavy-atom derivatives, dl and d2- We assume that we have been able to detemiine the heavy-atom positions in the derivatives and hl and h2 are the calculated heavy-atom contributions to the structure amplitudes of the derivatives. The centres of the derivative circles are at points - hl and - h2 in the complex plane, and the three circles intersect at one point, which is therefore the complex value of The phases for as many reflections as possible can then be... 

Figure Bl.8.5. Pp Pdl and Fdl are the measured stnicture amplitudes of a reflection from a native protein and from two heavy-atom derivatives. and are the heavy atom contributions. The pomt at which the tliree circles intersect is the complex value of F. ...

Figure 18.10 The diffracted waves from the protein part (ted) and from the heavy metals (green) interfere with each other in crystals of a heavy-atom derivative. If this interference is positive as illustrated in (a), the intensity of the spot from the heavy-atom derivative (blue) crystal will be stronger than that of the protein (red) alone (larger amplitude). If the interference is negative as in (b). the reverse is true (smaller amplitude).

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... 

However it would be a great mistake to believe that all actions of metal drugs will be at the DNA level. The metal complexes described here act in a highly selective manner with proteins — this is why they are used to provide heavy atom derivatives for crystallographic work. Thus we may expect that there will be other effects of the heavy metals which are associated with RNA and protein interactions. 

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... 

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 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. 

NCP crystals. There were two facets to this approach. First, it was necessary to reconstitute NCPs from a defined sequence DNA that phased precisely on the histone core to circumvent the random sequence disorder. It was obvious that the DNA was important for the quality of the diffraction from NCP crystals but the role of histone heterogeneity was not so clear. Heavy atom derivatives (i.e., electron rich elements bound in specific positions on the proteins) were not readily prepared by standard soaking experiments, due to a paucity of binding sites. Hence, it was necessary to selectively mutate amino acid residues in the histones to create binding sites for heavy atoms. 

Figure 6.1 The Periodic table showing the elements used successfully as heavy-atom derivatives in bold and underlined. The rest of the elements are shown only for completeness.

Figure 6.2 Vector (Argand) diagram showing the reiationships between heavy-atom derivative (Fpn), native protein (Fp) and heavy atom (Fpi) ap is the phase angie for the native protein. The vectors are piotted in the compiex piane.

From Eq. 3 and Fig. 6.3a it is clear that with only one heavy-atom derivative (single isomor-phous replacement SIR) the resultant phase will have two values (ap and apb) one of these phases will represent that of one structure and the other of its mirror image. But, since proteins contain only L-amino acids, this phase ambiguity must be eliminated using a second derivative, the anomalous component of the heavy atom or by solvent levelling (Wang, 1985), as shown diagrammatically in Fig. 6.3b. 

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.

Dr Bart Hazes (University of Alberta) has further grouped heavy-atom derivatives in six different categories as follow ... 

As indicated above, the search for suitable heavy-atom derivatives is as empirical as searching for crystallization conditions. To speed up the process and to save time, initial scanning for suitable heavy-atom derivatives can be done visually, using small... 

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. 

Hatfull, G. R, Sanderson, M. R., Freemont, P. S., Raccuia, P. R., Grindley, N. D. F. and Steitz, T. A. (1989). Preparation of heavy-atom derivatives using site-directed mutagenesis introduction of cysteine residues into yS resolvase. /. Mol. Biol. 208,661-667. 

Holbrook, S. R. and Kim, S.-H. (1985). CrystalUzation and heavy-atom derivatives of polynucleotides. Method Enzymol. 114,167-176. 

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

If, for some reason, MR failed, leaving no option but to look for heavy atom derivatives and if, for lack of isomorphism, the phasing power of these... 

Fig. 9. Approximate Bragg resolution for the first exposure of each of about 200 crystals from H. marismortui SOS subunits that were investigated at XI1, EMBL/DESY, Hamburg (ERG), in August 1986 at —4° to 19 °C. Shading indicates heavy-atom derivative test crystals (undecagold-cluster and tetrakis(acetoxymercuri)methane (TAMM) see paragraph 4 Phase Determination)...

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). 

Since protein BLl 1 is nearly globular its location may be determined in a Patterson map with coefficients of [F(wild)-F(mutant)] and may serve, by itself, as a giant heavy-atom derivative. At preliminary stages of structure determination this approach may provide phase information and reveal the location of the lacking protein. 

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